Vista regulatory t cell mediator protein, vista binding agents and use thereof

ABSTRACT

The present invention relates to a novel regulatory T cell protein. This protein, designated PD-L3 OR VISTA resembles members of the PD-L1 family, identified a novel and structurally-distinct, Ig-superfamily inhibitory ligand, whose extracellular domain bears homology to the B7 family ligand PD-L1. This molecule is designated as PD-L3 OR VISTA or V-domain Immunoglobulin Suppressor of T cell Activation (VISTA). Expression of VISTA is primarily within the hematopoietic compartment and is highly regulated on myeloid APCs and T cells. Therapeutic intervention of the VISTA inhibitory pathway represents a novel approach to modulate T cell-mediated immunity for the treatment of a wide variety of cancers, e.g., ovarian, bladder cancer and melanomas. Also, VISTA proteins, especially multimeric VISTA proteins and antibodies may be used to suppress T cell immunity in autoimmune disease, allergy, infection and inflammatory conditions, e.g. multiple sclerosis and artritic conditions such as RA.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/467,118 filed Mar. 23, 2017, now U.S. Pat. No. 10,781,254, which is adivisional of U.S. patent application Ser. No. 13/637,381, which has a371(c) date of Apr. 2, 2013, now U.S. Pat. No. 9,631,018, which is aU.S. National Stage entry of International Pat. Appl. No.PCT/US2011/030087, filed Mar. 25, 2011, which is a continuation of U.S.application Ser. No. 12/732,371 filed Mar. 26, 2010, now U.S. Pat. No.8,231,872, which claims priority to U.S. Provisional Appl. No.61/390,434 filed Oct. 6, 2010, U.S. Provisional Appl. No. 61/436,379filed Jan. 26, 2011 and U.S. Provisional Appl. No. 61/449,882 filed Mar.7, 2011, each of which is hereby incorporated by reference in itsentirety.

This invention was made with government support under Grant #ROIA1048667 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INTRODUCTION Sequence Disclosure

This application includes as part of its disclosure a biologicalsequence listing which is being concurrently submitted through EFS-Web.Said biological sequence listing is contained in a file named“11432520001087.txt” which was created Sep. 17, 2020, and has a size of27,761 bytes, and is hereby incorporated by reference in its entirety.

This application relates to We have discovered, characterized andfunctionally defined a novel, hematopoietically-restricted,structurally-distinct, Ig-superfamily inhibitory ligand designated asV-region Immunoglobulin-containing Suppressor of T cell Activation(VISTA) or PD-L3. The extracellular domain bears homology to the B7family ligand PD-L1, and like PD-L1, VISTA has a profound impact onimmunity. However, unlike PD-LI, expression of VISTA is exclusivelywithin the hematopoietic compartment. Expression is most prominent onmyeloid antigen-presenting cells (APCs), although expression on CD4+ Tcells and on a subset of Foxp3+ regulatory T cells (Treg) is also ofsignificant interest. A soluble VISTA-1g fusion protein, or VISTAexpression on APCs, potently inhibits in vitro T cell proliferation,cytokine production and induces Foxp3 expression in T cells. Conversely,a newly developed anti-VISTA monoclonal antibody interfered withVISTA-induced-immune suppression of T cell responses by VISTA+ APCs invitro. Furthermore, in vivo anti-VISTA intensified the development ofthe T cell mediated autoimmune disease experimental allergicencephalomyelitis (EAE), and facilitated the development of aprotective, tumor-specific immune response with subsequent tumorremission. Initial studies of VISTA−/− mice are revealing earlyindications of spontaneous inflammatory disease, and their ultimatepathologic fate will be determined. Unlike all other PD-Ligand-relatedmolecules (e.g., B7-H3, H4, H6), the hematopoietic restriction of VISTAtogether with its profound suppressive activities and unique structuralfeatures, illustrates that VISTA is a novel, functionally non-redundant.central negative regulator of immunity, whose expression is primarilymyeloid-restricted.

BACKGROUND OF THE INVENTION

Induction of an immune response requires T cell expansion,differentiation, contraction and establishment of T cell memory. T cellsmust encounter antigen presenting cells (APCs) and communicate via Tcell receptor (TCR)/major histocompatibility complex (MHC) interactionson APCs. Once the TCR/MHC interaction is established, other sets ofreceptor-ligand contacts between the T cell and the APC are required,i.e. co-stimulation via CD154/CD40 and CD28/B7.1-B7.2. The synergybetween these contacts is suggested to result, in vivo, in a productiveimmune response capable of clearing pathogens and tumors, and in somecases capable of inducing autoimmunity.

Another level of control has been identified, namely regulatory T cells(Treg). This specific subset of T cells is generated in the thymus,delivered into the periphery, and is capable of constant and induciblecontrol of T cells responses in vitro and in vivo (Sakaguchi (2000) Cell101(5):455-8; Shevach (2000) Annu. Rev. Immunol. 18:423-49; Bluestoneand Abbas (2003) Nat. Rev. Immunol. 3(3):253-7). Treg are represented bya CD4+CD25+ phenotype and also express high levels of cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4), OX-40, 4-1BB and theglucocorticoid inducible TNF receptor-associated protein (GITR)(McHugh,et al. (2002) Immunity 16(2):311-23; Shimizu, et al. (2002) Nat. Immun.3(2):135-42). Elimination of Treg cells by 5 day neonatal thymectomy orantibody depletion using anti-CD25, results in the induction ofautoimmune pathology and exacerbation of T cells responses to foreignand self-antigens, including heightened anti-tumor responses (Sakaguchi,et al. (1985) J. Exp. Med. 161(1):72-87; Sakaguchi, et al. (1995) J.Immunol. 155(3):1151-64; Jones, et al. (2002) Cancer Immun. 2:1). Inaddition, Treg have also been involved in the induction and maintenanceof transplantation tolerance (Hara, et al. (2001) J. Inununol.166(6):3789-3796; Wood and Sakaguchi (2003) Nat. Rev. Immunol.3:199-210), since depletion of Treg with anti-CD25 monoclonal antibodiesresults in ablation of transplantation tolerance and rapid graftrejection (Jarvinen, et al. (2003) Transplantation 76:1375-9). Among thereceptors expressed by Treg, GITR seems to be an important componentsince in vitro or in vivo ligation of GITR on the surface of Treg withan agonistic monoclonal antibody results in rapid termination of Tregactivity (McHugh, et al. (2002) supra; Shimizu, et al. (2002) supra),also resulting in autoimmune pathology (Shimizu, et al. (2002) supra)and ablation of transplantation tolerance.

Costimulatory and co-inhibitory ligands and receptors not only provide a“2nd signal” for T cell activation, but also a balanced network ofpositive and negative signal to maximize immune responses againstinfection while limiting immunity to self. The best characterizedcostimulatory ligands are B7.1 and B7.2, which are expressed byprofessional APCs, and whose receptors are CD28 and CTLA-4 (Greenwald,R. J., Freeman, G. J., and Sharpe, A. H. (2005). Annu Rev Immunol 23,515-548; Sharpe, A. H., and Freeman, G. J. (2002) Nat Rev Immunol 2,116-126). CD28 is expressed by naïve and activated T cells and iscritical for optimal T cell activation. In contrast, CTLA-4 is inducedupon T cell activation and inhibits T cell activation by binding toB7.1/B7.2, thus impairing CD28-mediated costimulation. CTLA-4 alsotransduces negative signaling through its cytoplasmic ITIM motif (Teft,W. A., Kirchhof, M. G., and Madrenas, J. (2006). Annu Rev Immunol 24,65-97; Teft, W. A., Kirchhof, M. G., and Madrenas, J. (2006). Annu RevImmunol 24, 65-97. B7.1/B7.2 KO mice are impaired in adaptive immuneresponse Borriello, F., Sethna, M. P., Boyd, S. D., Schweitzer, A. N.,Tivol, E. A., Jacoby, D., Strom, T. B., Simpson, E. M., Freeman, G. J.,and Sharpe, A. H. (1997)) Immunity 6, 303-313; Freeman, G. J.,Borriello, F., Hodes, R. J., Reiser, H., Hathcock, K. S., Laszlo, G.,McKnight, A. J., Kim, J., Du, L., Lombard, D. B., and et al. (1993).Science 262, 907-909), whereas CTLA-4 KO mice mice can not adequatelycontrol inflammation and develop systemic autoimmune diseases (Chambers,C. A., Sullivan, T. J., and Allison, J. P. (1997) Immunity 7, 885-895;Tivol, E. A., Borriello, F., Schweitzer, A. N., Lynch, W. P., Bluestone,J. A., and Sharpe, A. H. (1995) Immunity 3, 541-547; Waterhouse, P.,Penninger, J. M., Timms, E., Wakeham, A., Shahinian, A., Lee, K. P.,Thompson, C B., Griesser, H., and Mak, T. W. (1995). Science 270,985-988.

The B7 family ligands have expanded to include costimulatory B7-H2 (ICOSLigand) and B7-H3, as well as co-inhibitory B7-H1 (PD-L1), B7-DC(PD-L2), B7-H4 (B7S1 or B7x), and B7-H6 Brandt, C. S., Baratin, M., Yi,E. C., Kennedy, J., Gao, Z., Fox, B., Haldeman, B., Ostrander, C. D.,Kaifu, T., Chabannon, C., et al. (2009) J Exp Med 206, 1495-1503;Greenwald, R. J., Freeman, G. J., and Sharpe, A. H. (2005) Annu RevImmunol 23, 515-548.

Inducible costimulatory (ICOS) molecule is expressed on activated Tcells and binds to B7-H2 Yoshinaga, S. K., Whoriskey, J. S., Khare, S.D., Sarmiento, U., Guo, J., Horan, T., Shih, G., Zhang, M., Coccia, M.A., Kohno, T., et al. (1999). Nature 402, 827-832. ICOS is important forT cell activation, differentiation and function, as well as essentialfor T-helper-cell-induced B cell activation, Ig class switching, andgerminal center (GC) formation Dong, C., Juedes, A. E., Temann, U. A.,Shresta, S., Allison, J. P., Ruddle, N. H., and Flavell, R. A. (2001)Nature 409, 97-101; Tafuri, A., Shahinian, A., Bladt, F., Yoshinaga, S.K., Jordana, M., Wakeham, A., Boucher, L. M., Bouchard, D., Chan, V. S.,Duncan, G., et al. (2001) Nature 409, 105-109; Yoshinaga, S. K.,Whoriskey, J. S., Khare, S. D., Sarmiento, U., Guo, J., Horan, T., Shih,G., Zhang, M., Coccia, M. A., Kohno, T., et al. (1999) Nature 402,827-832. Programmed Death 1 (PD-1) on the other hand, negativelyregulates T cell responses. PD-1 KO mice develop lupus-like autoimmunedisease, or autoimmune dilated cardiomyopathy depending upon the geneticbackground Nishimura, H., Nose, M., Hiai, H., Minato, N., and Honjo, T.(1999) Immunity 11, 141-151. Nishimura, H., Okazaki, T., Tanaka, Y.,Nakatani, K., Hara, M., Matsumori, A., Sasayama, S., Mizoguchi, A.,Hiai, H., Minato, N., and Honjo, T. (2001) Science 291, 319-322. Theautoimmunity most likely results from the loss of signaling by bothligands PD-L1 and PD-L2. Recently, CD80 was identified as a secondreceptor for PD-L1 that transduces inhibitory signals into T cellsButte, M. J., Keir, M. E., Phamduy, T. B., Sharpe, A. H., and Freeman,G. J. (2007) Immunity 27, 111-122. The receptor for B7-H3 and B7-H4still remain unknown.

The best characterized costimulatory ligands are B7.1 and B7.2 and theybelong to the Ig superfamily which consists of many critical immuneregulators, such as the B7 family ligands and receptors. Ig superfamilymembers are expressed on professional antigen-presenting cells (APCs),and their receptors are CD28 and CTLA-4. CD28 is expressed by naïve andactivated T cells and is critical for optimal T-cell activation. Incontrast, CTLA-4 is induced following T-cell activation and inhibitsT-cell activation by binding to B7.1/B7.2, impairing CD28-mediatedcostimulation. B7.1 and B7.2 knockout (K0) mice are impaired in adaptiveimmune response, whereas CTLA-4 KO mice cannot adequately controlinflammation and develop systemic autoimmune diseases. Over time the B7family ligands have expanded to include costimulatory ligands such asB7-H2 (ICOS Ligand) and B7-H3, and coinhibitory ligands such as B7-H1(PD-L1), B7-DC (PD-L2), B7-H4 (B7S1 or B7x), and B7-H6. Accordingly,additional CD28 family receptors have been identified. ICOS is expressedon activated T cells and binds to B7-H2. ICOS is a positiveco-regulator, important for T-cell activation, differentiation andfunction. On the other hand, programmed death 1 (PD-1) negativelyregulates T cell responses. PD-1 KO mice developed lupus-like autoimmunedisease, or t dilated cardiomyopathy. In contrast to VISTA (theimmunosupressive molecule which is the focus of present invention), thetwo inhibitory B7 family ligands, PD-L1 and PD-L2, have distinctexpression patterns. PD-L2 is inducibly expressed on DCs andmacrophages, whereas PD-L1 is broadly expressed on both hematopoieticcells and nonhematopoietic cell types. Consistent with theimmune-suppressive role of PD-1 receptor, studies using PD-L1−/− andPD-L2−/− mice have shown that both ligands have overlapping roles ininhibiting T-cell proliferation and cytokine production. PD-L1deficiency enhances disease progression in both the non-obese diabetic(NOD) model of autoimmune diabetes and the murine model of multiplesclerosis (experimental autoimmune encephalomyelitis; EAE). PD-L1−/− Tcells produce elevated levels of the proinflammatory cytokines in bothdisease models. In addition, studies in NOD mice have demonstrated thatthe tissue expression of PD-L1 (i.e. within pancreas) uniquelycontributes to its capacity of regionally controlling inflammation.PD-L1 is also highly expressed on placental syncytiotrophoblasts, whichcritically control the maternal immune responses to allogeneic fetus.

Given the powerful impact of this family of molecules on regulatingimmunity, substantial efforts in murine models of cancer have shown thatprotective anti-umor immunity can be induced by targeting this family ofmolecules. Studies involving anti-CTLA-4 have documented enhancedtherapeutic benefit in murine models and clinical trials of melanoma.Mice vaccinated with B16-GM-CSF (Gvax) promote the rejection of B16melanomas when combined with antibody blockade of CTLA-4. Antibodies toPD-1 as well as PD-L1 also document enhanced anti-tumor immunity andhost survival in a wide range of murine tumor models. Finally, althoughCTLA-4 and PD-1 belong to the same family of co-inhibitory molecules,evidence suggests they use distinct nonredundant mechanisms to inhibitT-cell activation, and there is synergy in the ability of anti-CTLA-4and anti-PD-1/L1 to enhance host survival in murine melanoma when usedin combination.

Based on the foregoing, the elucidation of another novel B7 type familymember and ligands and modulators thereof would be useful given theimportant role of these family members in regulating immunity,especially T cell immunity.

SUMMARY OF THE INVENTION

The present invention relates to therapeutic methods that modulate theactivity and/or which specifically bind or block the binding of aspecific regulatory T cell protein to its counterreceptor. This protein,designated PD-L3 OR VISTA, is a novel and structurally-distinct,Ig-superfamily inhibitory ligand, whose extracellular domain bearshomology to the B7 family ligand PD-L1. This molecule is referred tointerchangeably herein as PD-L3 or VISTA or as V-domain ImmunoglobulinSuppressor of T cell Activation (VISTA). VISTA is expressed primarilywithin the hematopoietic compartment and is highly regulated on myeloidAPCs and T cells. Therapeutic intervention of the VISTA inhibitorypathway represents a novel approach to modulate T cell-mediated immunityfor the treatment of a wide variety of cancers.

The present invention in particular relates to the use of antibodiesspecific to VISTA or PD-L3 to treat specific cancers including bladdercancer, ovarian cancer, and melanoma.

In addition, the present invention in particular relates to the use pfPD-L3 or VISTA proteins, especially multimeric VISTA proteins and viralvectors (e.g., adenoviral) that express same to treat conditions whereinimmunosupression is therapeutically desired such as allergy,autoimmunity and inflammatory conditions.

As disclosed infra, the expression of VISTA appears to be exclusive tothe hematopoietic compartment and this protein is highly expressed onmature myeloid cells (CD11b^(bright)), with lower levels of expressionon CD4⁺ T cells, T^(reg) and CD8⁺ T cells. Soluble VISTA proteins, e.g.,soluble VISTA-Ig fusion protein, or VISTA expression on APCs, suppressesin vitro CD4⁺ and CD8⁺ T cell proliferation and cytokine production. Itis also observed that anti-VISTA antibodies, e.g., an anti-VISTA mab(13F3) blocked VISTA-induced suppression of T cell responses by VISTA⁺APCs in vitro. Also, it has been discovered that an anti-VISTA mabexacerbated EAE and increased the frequency of encephalitogenic Th17s invivo. Still further, as disclosed in detail infra, it has been foundthat an anti-VISTA mab induces tumor remission in multiple (4) murinetumor models. VISTA expression on myeloid derived suppressor cells(MDSC) in these models is extremely high, suggesting that VISTA⁺ MDSCsuppress tumor specific immunity. As shown herein, VISTA exertsimmunosuppressive activities on T cells both in vitro and in vivo, inmouse and in human (in vitro only) and is an important mediator incontrolling the development of autoimmunity and the immune responses tocancer. Specifically, the data show that:

(1) VISTA is a new member of the Ig superfamily and contains an Ig-Vdomain with distant sequence similarity to PD-L1. We disclose hereinthat when produced as an Ig fusion protein or when overexpressed onartificial APCs VISTA inhibits both mouse and human CD4+ and CD8+ T cellproliferation and cytokine production.

(2) VISTA expression on myeloid APCs is inhibitory for T cell responsesin vitro.

VISTA expression on MDSC in the tumor microenvironment is extremelyhigh. Phenotypic and functional analysis of many cell surface moleculespreviously suggested to be involved in MDSC-mediated suppression of Tcells: CD115, CD124, CD80, PD-L1, and PD-L2 were expressed by MDSC butwith no differences in the levels of their expression or proportion ofpositive cells were found between MDSC and cells from tumor-free micethat lack immune suppressive activity. Therefore, we predict that VISTAwill be the primary B7 negative regulator on MDSCs.

(4) Antibody-mediated VISTA blockade induces protective immunity to anautologous tumor.

Based thereon, VISTA appears to be a dominant, negative immuneregulatory molecule on MDSCs that interferes with the development ofprotective anti-tumor immunity. Therefore, blocking the activity of thismolecule with anti-VISTA antibodies will permit the development ofprotective anti-tumor immunity in humans and other mammals.

Therefore, the invention relates to methods of using soluble VISTAproteins, e.g., fusion proteins and multimeric VISTA proteins comprisingmultiple copies of the VISTA extravcelular domain or a fragment thereof,and VISTA binding agents, e.g., small molecules and antibodies orfragments theeof, which bind or modulate (agonize or antagonize) theactivity of VISTA as immune modulators and for the treatment ofdifferent cancers, e.g., bladder, ovarian and lymphoma, autoimmunedisease, allergy, infection and inflammatory conditions, e.g. multiplesclerosis and arthritis.

As described in detail infra, this protein is a novel inhibitory ligand,which extracellular Ig-V domain bears homology to the two known B7family ligands Programmed Death Ligand 1 and 2 (PD-L1 and PD-L2) andexhibits unique sequence features and distinctive expression patterns invitro and in vivo on subsets of APCs and T cells, (which distinguishesPD-L3 or VISTA from other B7 family ligands). This protein has beenshown to have a functional impact on CD4⁺ and CD8⁺ T cell proliferationand differentiation (suppresses CD4⁺ and CD8⁺ T cell proliferation, aswell as cytokine production). Based on its expression pattern andinhibitory impact on T cells, PD-L3 OR VISTA apparently functions as aregulatory ligand that negatively regulates T cell responses duringcognate interactions between T cells and myeloid derived APCs.

While PD-L3 OR VISTA appears to be a member of the B7 family of ligands,unlike other B7 family ligands, this molecule contains only an Ig-Vdomain without an Ig-C domain, and is phylogenically closer to the B7family receptor Programmed Death-1 (PD-1). Based thereon, PD-L3 ORVISTA, and agonists or antagonists specific thereto can be used toregulate T cell activation and differentiation, and more broadly tomodulate the regulatory network that controls immune responses. Inparticular PD-L3 OR VISTA proteins and PD-L3 OR VISTA agonists orantagonists, preferably antibodies specific to PD-L3 OR VISTA are usefulin modulating immune responses in autoimmunity, inflammatory responsesand diseases, allergy, cancer, infectious disease and transplantation.

Therefore, the present invention in part relates to compositions e.g.,for therapeutic, diagnostic or immune modulatory usage containing anisolated soluble PD-L3 OR VISTA protein or fusion protein, e.g., asoluble VISTA-Ig fusion protein or a multimeric VISTA protein,comprising an amino acid sequence that preferably is at least 70-90%identical to the human or murine PD-L3 OR VISTA polypeptide set forth inSEQ ID NO:2, 4 or 5 or an ortholog, or fragment thereof encoded by agene that specifically hybridizes to SEQ ID NO:1 or 3 that modulatesVISTA in vivo and a pharmaceutically acceptable carrier. In someembodiments, the soluble or multimeric VISTA protein may be directly orindirectly linked to a heterologous (non-VISTA) protein or may beexpressed by a viral vector or a cell containing, e.g., a transfectedimmune cell such as a T cell.

The present invention also provides expression vectors comprising anisolated nucleic acid encoding a VISTA protein that is at least 70-90%identical to the human or murine VISTA amino acid sequence set forth inSEQ ID NO:2, 4 or 5 or a fragment or ortholog thereof, which optionallyis fused to a sequence encoding another protein such as an Igpolypeptide, e.g., an Fc region or a reporter molecule; and host cellscontaining said vectors.

The present invention also specifically relates to an isolated bindingagent, preferably an antibody or antibody fragment which specificallybinds to a PD-L3 OR VISTA protein comprising the amino acid sequence setforth in SEQ ID NO:2, 4 or 5 or a variant, fragment or ortholog thereof.In a preferred embodiment, the binding agent modulates (agonizes orantagonizes) VISTA activity in vitro or in vivo. In most preferredembodiments, the binding agent is an agonistic or antagonistic antibody.

The present invention further provides methods for modulating an immunecell response by contacting an immune cell in vitro or in vivo with aVISTA protein, or binding agent specific thereto, in the presence of aprimary signal so that a response of the immune cell is modulated.(Interaction of r VISTA or a modulator thereof transmits a signal toimmune cells, regulating immune responses. PD-L3 OR VISTA protein isexpressed at high levels on myeloid antigen presenting cells, includingmyeloid dendritic cells (DCs) and macrophages, and at lower densities onCD4+ and CD8+ T cells. Upon immune activation, PD-L3 or VISTA expressionis upregulated on myeloid APCs, but downregulated on CD4+ T cells).Therefore, the PD-L3 or VISTA nucleic acids and polypeptides of thepresent invention, and agonists or antagonists thereof are useful, e.g.,in modulating the immune response.

In another aspect this invention provides isolated nucleic acidmolecules encoding VISTA polypeptides, preferably encoding solublefusion proteins and multimeric VISTA proteins as well as nucleic acidfragments suitable as primers or hybridization probes for the detectionof PD-L3 or VISTA-encoding nucleic acids. In one embodiment, a PD-L3 orVISTA nucleic acid molecule of the invention is at least about 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the nucleotide sequence (e.g., to the entire length of thenucleotide sequence) encoding PD-L3 or VISTA in SEQ ID NO:1 or 3 shownherein or a complement thereof.

In another embodiment, a PD-L3 or VISTA nucleic acid molecule includes anucleotide sequence encoding a polypeptide having an amino acid sequencehaving a specific percent identity to the amino acid sequence of SEQ IDNO: 2, 4 or 5. In a preferred embodiment, a PD-L3 or VISTA nucleic acidmolecule includes a nucleotide sequence encoding a polypeptide having anamino acid sequence at least about 71%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more identical to the entire length of the amino acidsequence of SEQ ID NO: 2, 4 or 5 or to the extracelllular domainthereof.

In another preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of human or murine or VISTA or aconserved region or functional domain therein. In yet another preferredembodiment, the nucleic acid molecule includes a nucleotide sequenceencoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4or 5. In yet another preferred embodiment, the nucleic acid molecule isat least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or morenucleotides in length. In a further preferred embodiment, the nucleicacid molecule is at least about 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150 or more nucleotides in length and encodes a polypeptide having aPD-L3 or VISTA activity or modulating PD-L3 or VISTA function (asdescribed herein).

Another embodiment of the invention features nucleic acid molecules,preferably PD-L3 or VISTA nucleic acid molecules, which specificallydetect PD-L3 or VISTA nucleic acid molecules relative to nucleic acidmolecules encoding non-PD-L3 or VISTA polypeptides. For example, in oneembodiment, such a nucleic acid molecule is at least about 880, 900,950, 1000, 1050, 1100, 1150 or more nucleotides in length and hybridizesunder stringent conditions to a nucleic acid molecule encoding thepolypeptide shown in SEQ ID NO: 2, 4 or 5, or a complement thereof. Inanother embodiment, such a nucleic acid molecule is at least 20, 30, 40,50, 100, 150, 200, 250, 300 or more nucleotides in length and hybridizesunder stringent conditions to a nucleic acid molecule encoding afragment of PD-L3 or VISTA, e.g., comprising n at least 20, 30, 40, 50,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950 or more nucleotides in length, includes at least 15(i.e., 15 contiguous) nucleotides of the disclosed nucleic acid sequencein SEQ ID NO: 1 and 3 encoding the PD-L3 or VISTA polypeptides in SEQ IDNO: 2, 4 or 5, or a complement thereof, and hybridizes under stringentconditions to a nucleic acid molecule comprising the nucleotide sequenceshown in SEQ ID NO: 1, or 3 or a complement thereof.

In still other preferred embodiments, the nucleic acid molecule encodesa naturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO: 2 or 4 or 5, wherein the nucleic acidmolecule hybridizes to a complement of a nucleic acid moleculecomprising SEQ ID NO: 1 or 3, or a complement thereof, under stringentconditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a PD-L3 or VISTA nucleic acid molecule,e.g., is antisense to the coding strand of a PD-L3 or VISTA nucleic acidmolecule as shown in SEQ ID NO: 1 or 3.

Another aspect of the invention provides a vector comprising a PD-L3 orVISTA nucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector.

In another embodiment, the invention provides a host cell containing avector of the invention. In yet another embodiment, the inventionprovides a host cell containing a nucleic acid molecule of theinvention. The invention also provides a method for producing apolypeptide, preferably a PD-L3 or VISTA polypeptide, by culturing in asuitable medium, a host cell, e.g., a mammalian host cell such as anon-human mammalian cell, of the invention containing a recombinantexpression vector, such that the polypeptide is produced.

Another aspect of this invention features isolated or recombinant PD-L3or VISTA polypeptides (e.g., proteins, polypeptides, peptides, orfragments or portions thereof). In one embodiment, an isolated PD-L3 orVISTA polypeptide or PD-L3 or VISTA fusion protein includes at least oneor more of the following domains: a signal peptide domain, an IgVdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

In a preferred embodiment, a PD-L3 or VISTA polypeptide includes atleast one or more of the following domains: a signal peptide domain, anIgV domain, an extracellular domain, a transmembrane domain, and acytoplasmic domain, and has an amino acid sequence at least about 71%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the amino acid sequence of SEQ ID NO: 2 or 4 or 5. Inanother preferred embodiment, a PD-L3 or VISTA polypeptide includes atleast one or more of the following domains: a signal peptide domain, anIgV domain, an extracellular domain, a transmembrane domain, and acytoplasmic domain, and has a VISTA or PD-L3 activity (as describedherein).

In yet another preferred embodiment, a PD-L3 polypeptide includes atleast one or more of the following domains: a signal peptide domain, anIgV domain, an extracellular domain, a transmembrane domain, and acytoplasmic domain, and is encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 1 or 3.

In another embodiment, the invention features fragments or portions ofthe polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4 or5, wherein the fragment comprises at least 15 amino acids (i.e.,contiguous amino acids) of the amino acid sequence of SEQ ID NO: 2 or 4.In another embodiment, a PD-L3 or VISTA polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 2, 4 or 5. In anotherembodiment, the invention features a PD-L3 or VISTA polypeptide which isencoded by a nucleic acid molecule consisting of a nucleotide sequenceat least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO: 1or 3, or a complement thereof. This invention further features a PD-L3or VISTA polypeptide which is encoded by a nucleic acid moleculeconsisting of a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 1 or 3.

The polypeptides of the present invention or portions thereof, e.g.,biologically active portions thereof, can be operatively linked to anon-PD-L3 or VISTA polypeptide (e.g., heterologous amino acid sequences)to form fusion polypeptides. The invention further features antibodies,such as monoclonal or polyclonal antibodies, that specifically bindpolypeptides of the invention, preferably human PD-L3 or VISTApolypeptides.

The invention also relates to methods of selecting anti-PD-L3 or VISTAantibodies having desired functional properties from panels ofmonoclonal antibodies produced against this protein or a PD-L3 orVISTA-Ig fusion protein based on desired functional properties, e.g.,modulating specific effects of PD-L3 or VISTA on immunity such as thesuppressive effect of the protein on TCR activation, the suppressiveeffect of the protein on CD4 T cell proliferative responses to anti-CD3,suppression of antigen specific proliferative responses of cognate CD4 Tcells, the suppressive effects of PD-L3 or VISTA on the expression ofspecific cytokines such as IL-2 and gamma interferon, et al. In aparticularly preferred embodiment anti-PD-L3 or VISTA antibodies for useas therapeutics will be selected that in vitro, in the presence ofsoluble PD-L3 or VISTA-proteins, e.g., PD-L3 or VISTA-Ig fusion proteinenhance the suppressive effects of PD-L3 or VISTA-Ig on PD-L3 or VISTArelated immune functions. This is preferred as quite unexpectedly (showninfra) these antibodies in vivo behave opposite to what would beexpected from their in vitro effect on immunity, i.e., these anti- orVISTA monoclonal antibodies are immunosuppressive.

In addition, the PD-L3 or VISTA polypeptides (or biologically activeportions thereof) or modulators of the PD-L3 or VISTA molecules, i.e.,antibodies such as selected using the foregoing methods can beincorporated into pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another embodiment, a PD-L3 or VISTA protein is used as an inhibitorysignal for inhibiting or decreasing immune cell activation. In thisembodiment, the inhibitory signal binds to an inhibitory receptor (e.g.,CTLA-4 or PD-1) on an immune cell thereby antagonizing the primarysignal which binds to an activating receptor (e.g., via a TCR, CD3, BCR,or Fc polypeptide). Inhibition includes, e.g., inhibition of secondmessenger generation; an inhibition of proliferation; an inhibition ofeffector function in the immune cell, e.g., reduced phagocytosis,reduced antibody production, reduced cellular cytotoxicity, the failureof the immune cell to produce mediators, (such as cytokines (e.g., IL-2)and/or mediators of allergic responses); or the development of anergy.

In particular embodiments, the primary signal is a ligand (e.g., CD3 oranti-CD3) that binds TCR and initiates a primary stimulation signal.Such TCR ligands are readily available from commercial sources andspecific examples include anti-CD3 antibody OKT3, prepared fromhybridoma cells obtained from the American Type Culture Collection, andanti-CD3 monoclonal antibody G19-4. In an alternative embodiment, aprimary signal is delivered to a T cell through other mechanismsincluding a protein kinase C activator, such as a phorbol ester (e.g.,phorbol myristate acetate), and a calcium ionophore (e.g., ionomycin,which raises cytoplasmic calcium concentrations), or the like. The useof such agents bypasses the TCR/CD3 complex but delivers a stimulatorysignal to T cells. Other agents acting as primary signals can includenatural and synthetic ligands. A natural ligand can include MHC with orwithout a peptide presented. Other ligands can include, but are notlimited to, a peptide, polypeptide, growth factor, cytokine, chemokine,glycopeptide, soluble receptor, steroid, hormone, mitogen, such as PHA,or other superantigens, peptide-MHC tetramers (Altman, et al. (1996)Science 274(5284):94-6) and soluble MHC dimers (Dal Porto, et al (1993)Proc Natl. Acad. Sci. USA 90: 6671-5).

Immune cells activated in accordance with the method of the instantinvention can subsequently be expanded ex vivo and used in the treatmentand prevention of a variety of diseases; e.g., human T cells which havebeen cloned and expanded in vitro maintain their regulatory activity(Groux, et al. (1997) Nature 389(6652):737-42). Prior to expansion, asource of T cells is obtained from a subject (e.g., a mammals such as ahuman, dog, cat, mouse, rat, or transgenic species thereof). T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, spleen tissue, tumors or T celllines. T cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation.

In another aspect, the present invention provides a method for detectingthe presence of a PD-L3 or VISTA nucleic acid molecule, protein, orpolypeptide in a biological sample by contacting the biological samplewith an agent capable of detecting a PD-L3 OR VISTA nucleic acidmolecule, protein, or polypeptide, such that the presence of a PD-L3 ORVISTA nucleic acid molecule, protein or polypeptide is detected in thebiological sample. This PD-L3 OR VISTA expression can be used to detectcertain disease sites such as inflammatory sites.

In another aspect, the present invention provides a method for detectingthe presence of PD-L3 OR VISTA activity in a biological sample bycontacting the biological sample with an agent capable of detecting anindicator of PD-L3 OR VISTA activity, such that the presence of PD-L3 ORVISTA activity is detected in the biological sample.

In another aspect, the invention provides a method for modulating PD-L3OR VISTA activity, comprising contacting a cell capable of expressingPD-L3 OR VISTA with an agent that modulates PD-L3 OR VISTA activity,preferably an anti-PD-L3 OR VISTA antibody such that PD-L3 OR VISTAactivity in the cell is modulated. In one embodiment, the agent inhibitsPD-L3 OR VISTA activity. In another embodiment, the agent stimulatesPD-L3 OR VISTA activity. In a further embodiment, the agent interfereswith or enhances the interaction between a PD-L3 OR VISTA polypeptideand its natural binding partner(s). In one embodiment, the agent is anantibody that specifically binds to a PD-L3 OR VISTA polypeptide. Inanother embodiment, the agent is a peptide, peptidomimetic, or othersmall molecule that binds to a PD-L3 OR VISTA polypeptide.

n still another embodiment, the agent modulates expression of PD-L3 ORVISTA by modulating transcription of a PD-L3 OR VISTA gene, translationof a PD-L3 OR VISTA mRNA, or post-translational modification of a PD-L3OR VISTA polypeptide. In another embodiment, the agent is a nucleic acidmolecule having a nucleotide sequence that is antisense to the codingstrand of a PD-L3 OR VISTA mRNA or a PD-L3 OR VISTA gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder or condition characterized byaberrant, insufficient, or unwanted PD-L3 OR VISTA polypeptide ornucleic acid expression or activity by administering an agent which is aPD-L3 OR VISTA modulator to the subject. In one preferred embodiment,the PD-L3 OR VISTA modulator is a PD-L3 OR VISTA polypeptide, preferablya soluble fusion protein or multimeric VISTA protein or anti-VISTAantibody as described infra. In another embodiment the PD-L3 OR VISTAmodulator is a PD-L3 OR VISTA nucleic acid molecule, e.g in anadenoviral vector. In another embodiment, the invention further providestreating the subject with an additional agent that modulates an immuneresponse.

In still another embodiment, the invention provides a vaccine comprisingan antigen and an agent that modulates (enhances or inhibits) PD-L3 ORVISTA activity. In a preferred embodiment, the vaccine inhibits theinteraction between PD-L3 OR VISTA and its natural binding partner(s).

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aPD-L3 OR VISTA polypeptide; (ii) misregulation of the gene; and (iii)aberrant post-translational modification of a PD-L3 OR VISTApolypeptide, wherein a wild-type form of the gene encodes a polypeptidewith a PD-L3 OR VISTA activity.

In another aspect the invention provides methods for identifying acompound that binds to or modulates the activity of a PD-L3 OR VISTApolypeptide, by providing an indicator composition comprising a PD-L3 ORVISTA polypeptide having PD-L3 OR VISTA activity, contacting theindicator composition with a test compound, and determining the effectof the test compound on PD-L3 OR VISTA activity in the indicatorcomposition to identify a compound that modulates the activity of aPD-L3 OR VISTA polypeptide.

In one aspect, the invention features a method for modulating theinteraction of PD-L3 OR VISTA with its natural binding partner(s) on animmune cell comprising contacting an antigen presenting cell whichexpresses PD-L3 OR VISTA with an agent selected from the groupconsisting of: a form of PD-L3 OR VISTA, or an agent that modulates theinteraction of PD-L3 OR VISTA and its natural binding partner(s) suchthat the interaction of PD-L3 OR VISTA with it natural bindingpartner(s) on an immune cell is modulated. In a preferred embodiment, anagent that modulates the interaction of PD-L3 OR VISTA and its naturalbinding partner(s) is an antibody that specifically binds to PD-L3 ORVISTA. In one embodiment, the interaction of PD-L3 OR VISTA with itsnatural binding partner(s) is upregulated. In another embodiment, theinteraction of PD-L3 OR VISTA with its natural binding partner(s) isdownregulated. In one embodiment, the method further comprisescontacting the immune cell or the antigen presenting cell with anadditional agent that modulates an immune response.

In one embodiment, the step of contacting is performed in vitro. Inanother embodiment, the step of contacting is performed in vivo. In oneembodiment, the immune cell is selected from the group consisting of: aT cell, a monocyte, a macrophage, a dendritic cell, a B cell, and amyeloid cell.

In another aspect, the invention pertains to a method for inhibiting orincreasing activation in an immune cell comprising increasing orinhibiting the activity or expression of PD-L3 OR VISTA in a cell suchthat immune cell activation is inhibited or increased.

In yet another aspect, the invention pertains to a vaccine comprising anantigen and an agent that inhibits the interaction between PD-L3 ORVISTA and its natural binding partner(s).

In still another aspect, the invention pertains to a vaccine comprisingan antigen and an agent that promotes the interaction between PD-L3 ORVISTA and its natural binding partner(s).

In another aspect, the invention pertains to a method for treating asubject having a condition that would benefit from upregulation of animmune response comprising administering an agent that inhibits theinteraction between PD-L3 OR VISTA and its natural binding partner(s) onimmune cells of the subject such that a condition that would benefitfrom upregulation of an immune response is treated. In one preferredembodiment, the agent comprises a blocking antibody or a small moleculethat binds to PD-L3 OR VISTA and inhibits the interaction between PD-L3OR VISTA and its natural binding partner(s). In another embodiment, themethod further comprises administering a second agent that upregulatesan immune response to the subject. In another aspect, the inventionpertains to a method for treating a subject having a condition thatwould benefit from downregulation of an immune response comprisingadministering an agent that stimulates the interaction between PD-L3 ORVISTA and its natural binding partner(s) on cells of the subject suchthat a condition that would benefit from downregulation of an immuneresponse is treated.

For example the condition treated with the PD-L3 OR VISTA protein orbinding agents is selected from the group consisting of: a tumor, apathogenic infection, an inflammatory immune response or condition,preferably less pronounced inflammatory conditions, or animmunosuppressive disease. Specific examples include multiple sclerosis,thyroiditis, rheumatoid arthritis, diabetes type II and type I andcancers, both advanced and early forms, including metastatic cancerssuch as bladder cancer, ovarian cancer, melanoma, lung cancer, and othercancers wherein VISTA suppresses an effective anti-tumor response. Insome case the individual may be administered cells or a viral vectorthat express a nucleic acid that encodes an anti-VISTA antibody or VISTAfusion protein.

In one embodiment agent comprises an antibody or a small molecule thatstimulates the interaction between PD-L3 OR VISTA and its naturalbinding partner(s). In another embodiment, the method further comprisesadministering a second agent that downregulates an immune response tothe subject such as a PD-L!, PD-L2 or CTLA-4 fusion protein or antibodyspecific thereto.

Exemplary conditions treatable using PD-L3 OR VISTA proteins, bindingagents or PD-L3 OR VISTA antagonists or agonists according to theinvention include by way of example transplant, an allergy, infectiousdisease, cancer, and inflammatory or autoimmune disorders, e.g., aninflammatory immune disorder. Specific examples of the foregoing includetype 1 diabetes, multiple sclerosis, rheumatoid arthritis, psoriaticarthritis, systemic lupus erythematosis, rheumatic diseases, allergicdisorders, asthma, allergic rhinitis, skin disorders, gastrointestinaldisorders such as Crohn's disease and ulcerative colitis, transplantrejection, poststreptococcal and autoimmune renal failure, septic shock,systemic inflammatory response syndrome (SIRS), adult respiratorydistress syndrome (ARDS) and envenomation; autoinflammatory diseases aswell as degenerative bone and joint diseases including osteoarthritis,crystal arthritis and capsulitis and other arthropathies. Further, themethods and compositions can be used for treating tendonitis,ligamentitis and traumatic joint injury.

In another aspect, the invention pertains to a cell-based assay forscreening for compounds which modulate the activity of PD-L3 OR VISTAcomprising contacting a cell expressing a PD-L3 OR VISTA target moleculewith a test compound and determining the ability of the test compound tomodulate the activity of the PD-L3 OR VISTA target molecule

In still another aspect, the invention pertains to a cell-free assay forscreening for compounds which modulate the binding of PD-L3 OR VISTA toa target molecule comprising contacting a PD-L3 OR VISTA polypeptide orbiologically active portion thereof with a test compound and determiningthe ability of the test compound to bind to the PD-L3 OR VISTApolypeptide or biologically active portion thereof.

In another embodiment, the invention pertains to a method of identifyinga compound, e.g. an anti-PD-L3 OR VISTA antibody which modulates theeffect of PD-L3 OR VISTA on T cell activation or cytokine production ata first and second antigen concentration comprising contacting a T cellexpressing a PD-L3 OR VISTA target molecule with a test compound at afirst antigen concentration, determining the ability of the testcompound to modulate T cell proliferation or cytokine production at thefirst antigen concentration, contacting a T cell expressing a PD-L3 ORVISTA target molecule with the test compound at a second antigenconcentration, and determining the ability of the test compound tomodulate T cell proliferation or cytokine production at the secondantigen concentration, thereby identifying a compound which modulates Tcell activation or cytokine production at a first and second antigenconcentration.

In other specific embodiments panels of anti-PD-L3 OR VISTA antibodiesand PD-L3 OR VISTA proteins are screened to select those of whichinhibit or promote the effects of PD-L3 OR VISTA on CD4+ and CD8+ T celldifferentiation, proliferation and/or cytokine production in vitro or invivo.

In preferred embodiments the subject PD-L3 OR VISTA proteins, nucleiacids, and ligands specific to PD-L3 OR VISTA, preferably antibodieshaving desired effects on PD-L3 OR VISTA functions are used to treatconditions such a cancer, autoimmune diseases, allergy, inflammatorydisorders or infection and more specifically immune system disorderssuch as severe combined immunodeficiency, multiple sclerosis, systemiclupus erythematosus, type I diabetes mellitus, lymphoproliferativesyndrome, inflammatory bowel disease, allergies, asthma,graft-versus-host disease, and transplant rejection; immune responses toinfectious pathogens such as bacteria and viruses; and immune systemcancers such as lymphomas and leukemias)

DETAILED DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E. Sequence analysis. FIG. 1A. Full length amino acid sequenceof murine PO-L3 OR VISTA. Figure B. Amino acid sequence alignment ofextracellular 1g domains between murine PD-L3 OR VISTA and selected B7family ligands, including B7-HI (PD-L1), B7-DC (PD-L2), B7-H3, and97-H4. Figure C Alignment of PD-L3 OR VISTA Ig domain with B7 familyreceptors, including PD-I, CTLA-4, CD28, BTLA, and ICOS. Ig-v domain, “. . . ”; Ig-c domain, “ . . . ”. Alignment was performed using theMUSCLE algorithm (MUltiple Sequence Comparison by Log-Expectation). FIG.1D. Sequence identity (%) of the Ig-V domains between PD-L3 OR VISTA andother B7 family ligands and receptors is calculated using ClustalW2program. FIG. 1E. Sequence homology between human and murine PD-L3 ORVISTA. Identical residues are shaded in black. Highly conserved andsemi-conserved residues are shaded in dark and light shade of grayrespectively.

FIG. 2. Phylogenic analysis of mouse PD-L3 OR VISTA with other1mmunoglobulin (1g) superfamily members. Full-length sequence of mousePD-L3 OR VISTA and other 1g superfamily members, including CD28, CTLA-4,ICOS, BTLA, PD B7-H1 (PD-LI), 8 7-DC(PD-L2), B7-H2, B7-H3, B7-H4, B7-1,87-2, BTNL2, BTN3 A3, BTN2A2, and BTN1AI, were analyzed using PhyMLalgorithm (Phylogenetic Maximum Likelihood). Branch distances were shownat tree branch joints.

FIG. 3A-G. Tissue expression and hematopoietic cell expression patternsof PD·L3 OR VISTA. FIG. 3A. RT-PCR of full-lengthPD-L3 OR VISTA frommouse tissues. Lanes: (1) muscle (2) heart (3) eye (4) thymus (5) spleen(6) small intestine (7) kidney (8) liver (9) brain (10) mammary gland(11) lung (12) ovary (13) bone marrow. FIG. 3B. RT-PCR of full-lengthPD-L3 OR VISTA from purified hematopoietic cell types. Lanes (1)peritoneal macrophages (2) splenic CD1 1b+ monocytes (3) splenic CD1 1e+DCs (4) splenic CD4+ T cells (5) splenic CD8+ T cells (6) splenic Bcells. FIG. 3C-E. Flow cytometry analysis of PD·L3 OR VISTA expressionon splenic CD4+ and CD8+ T cells from thymus and spleen (FIG. 3C), onCD1 1b+ monocytes (FIG. 3D), and on CD11c+ DC subsets from spleen andperitoneal cavity (FIG. 3E). F. Splenic B cells, NK cells andgranulocytes are also analyzed. FIG. 3G. The differential expression ofPD-L3 OR VISTA on hematopoietic cells from different tissue sites.including mesenteric LN, peripheral LN. spleen, blood and peritonealcavity. Representative data from at least 3 independent experiments areshown,

FIGS. 4A-1, 4A-2, 4A-3, 4B, 4C-1, 4C-2, 4C-3 and 4D. Gene array data ofPD-L3 OR VISTA from the GNF (Genomics institute of Novartis ResearchFoundation) gene array database, as well as the NCBI GEO (geneexpression omnibus) database.

FIG. 5. Specificity of PD-L3 OR VISTA hamster monoclonal antibodies.Mouse EU cell lines over-expressing either PD-LI Or PD-L3 OR VISTA fusedto RFP were stained using the supernatants from hybridoma cultures andanalyzed by flow cytometry. Two representative positive clones areshown.

FIG. 6. Comparison of PD-L3 OR VISTA express ion with other B7 familyligands on in vitro cultured spleen cells. Expression of PD-L3 OR VISTAand other B7 family ligands (i.e. PD-LI. PD-L2, B7-H3, and B7-H4) onhematopoietic cell types, including CD4+ T cells, CD1 1bhi monocytes,and CD1 1c+ DCs were compared. Cells were either freshly isolated. or invitro cultured for 24 hrs, with and without activation. CD4+ T cellswere activated with plate-bound □CD3 (5 ug/ml), CD1 1bhi monocytes andCD1 1c+ DCs were activated with IFNalpha (20 ng/ml) and LPS (200 ng/ml).Representative results from three independent experiments are shown.

FIGS. 7A and B. Comparison of in vivo expression patterns of PD-L3 ORVISTA and other B7 family ligands during immunization. D011.10 TCRtransgenic mice were immunized with chicken ovalbumin (OVA) emulsifiedin complete Freund's adjuvant (CFA) on the flank. Draining andnon-draining lymph node cells were collected 24 hr post immunization,and analyzed by flow cytometry for the expression of PD-L3 OR VISTA,PD-LI and PD-L2. Shown are representative results from at leas t fourindependent experiments. FIG. 7A. A population of CD1 1b+ cellsexpressing a high level of PD-L3 OR VISTA was induced at 24 hr postimmunization with CFA/OVA, but not with CFA alone within the draininglymph node. These cells are of mixed phenotype of F4180+ macrophages andCD11C+ dendritic cells. FIG. 7B. Expression of PD-L3 OR VISTA, PD-LI andPD-L2 on CD1 Ibhi monocytes, CO1 1c+ DCs and CD4+ T cells were analyzedat 24 hr post immunization.

FIG. 8. Loss of PD-L3 OR VISTA express ion on activated CD4+ T cells inresponse to immunization. DO11. 10 mice were immunized with chickenovalbumin (OVA) emulsified in complete Freund's adjuvant (CFA) on theflank. Draining and non-draining lymph node cells were collected 48 hrpost immunization, and analyzed for PD-L3 OR VISTA expression by flowcytometry. Shown are representative results from 2 independentexperiments.

FIGS. 9A-D. Immobilized PD-L3 OR VISTA-Ig fusion protein inhibited CD4+and CD8+ T cell proliferation. FIG. 9A. CFSE labeled CD4+ and CD8+ Tcells were stimulated by plate-bound □CD3 with or without co-absorbedPD-L3 OR VISTA-1g. The percentage of CFSE-low cells was quantified andshown in FIG. 9B. FIG. 9C CD4+ T cells from PO-I ko mice were alsosuppressed by PD-L3 OR VISTA-1g. FIG. 9D. PD-L3 OR VISTA-Ig-mediatedsuppression is persistent and can act late. CD4+ T cells were activatedin the presence of PD-L3 OR VISTA-1g or control-Ig for either 721 μs(i). or for 24 hrs (ii, in and iv). 24 hr-preactivated cells wereharvested and re-stimulated under specified conditions for another 48hrs. Cell proliferation was analyzed at the end of the 72 hr culture.(ii) Pre-activation with PD-L3 OR VISTA-Ig and re-stimulation withantiCD3; (iii) Pre-activation with antiCD3 and re-stimulation with PD-L3OR VISTA-Jg. (iv) Pre-activation with PD-L3 OR VISTA-1g andre-stimulation with PD-L3 OR VISTA-1g. Duplicated wells were analyzedfor conditions. Shown are representative results from at least fourexperiments.

FIG. 10. Similar inhibitory effect of PD-L1-Ig and PD-L3 OR VISTA-Igfusion proteins on CD4+ T cell proliferation. Bulk purified CD4+ T cellswere CFSE labeled and stimulated with plate-bound iJCD3 together withtitrated amount of PD-LI·1g or PD-L3OR VISTA-1 g fusion proteins. CFSEdilution was analyzed at 72 hrs and the percentage of CFSElow cells wasquantified. Duplicated wells were analyzed for all conditions. Shown arerepresentative results from 2 independent experiments.

FIGS. 11A and B. Suppressive impact of PD-L3 OR VISTA-1g on theproliferation of naïve and memory CD4+ T cells. FIG. 11A. Naïve(CD25-CD44lowCD621.hi) and memory (CD25-CD44hiCD62Llow) CD4+ T cellsubsets were sorted, CFSE labeled, and stimulated with plate boundanti-CD3 (2.5 μg/ml) together with PD-L3 OR VISTA-1g or control-1g atindicated ratios. Cell proliferation was analyzed at 72 hrs by examiningthe CFSE division profile. The percentage of proliferated cells, asdetermined by percentage of CFSElow cells, is calculated and shown inFIG. 11B. Duplicated wells were analyzed for all conditions. Shown arerepresentative results from two independent experiments.

FIGS. 12A and B. PD-L3 OR VISTA-1g fusion protein suppressed early TCRactivation and cell proliferation, but did not directly induceapoptosis. Bulk purified CD4+ T cells were stimulated with plate-boundanti-CD3 together with PD-L3 OR VISTA-Ig or control-Ig at 1-2 ratio (2.5μg/ml and 5 μg/ml respectively). Cells were analyzed at 24 hr and 48 hrsfor the expression of CD69, CD62L, and CD44 by flow cytometry. Cellswere also stained for early apoptosis marker annexin-V, and cell deathmarker 7-Aminoactinomycin D (7-AAD). Shown are representative resultsfrom two independent experiments.

FIGS. 13A-E. PD-L3 OR VISTA-1g inhibited cytokine production by CD4+ andCD8+ T cells. FIGS. 13A-B. Bulk purified CD4+ T cells were stimulatedwith plate-bound anti-CD3, and PD-L3 OR VISTA-1g or control-1g al statedratios. Culture supernatants were collected after 24 hrs and 48 hrs.Levels of IL-2 and IFN□. were analyzed by ELISA. FIGS. 13C-D. CD4+ Tcells were sorted into na'ive (CD25-CD44IowCD621 . . . hi) and memory(CD25− CD44hiCD62Llow) cell populations. Cells were stimulated withplate-bound □CD3 and PD-L3 OR VISTA-1g or control-1g at a ratio of I:2.Culture supernatants were collected at 48 hrs and analyzed for the levelof i1 . . . −2 and IFN∘ by ELISA. FIG. 13E. Bulk purified CD8+ T cellswere stimulated with plate-bound □CD3, and PD-L3 OR VISTA-1g orcontrol-Ig at indicated ratios. IFN□ in the culture supernatant wasanalyzed by ELISA. For all conditions, supernatant for six duplicatedwells were pooled for ELBA analysis. Shown are representative resultsfront at least three experiments.

FIGS. 14A-D. PD-L3 OR VISTA-Ig-mediated suppression could overcome amoderate level of costimulation provided by CD28, but was completelyreversed by a high level of costimulation, as well as partially rescuedby exogenous IL-2. FIGS. 14A-B. CD4+ T cells were activated byplate-bound □CD3 together with either PD-L3 OR v iSTA-1g or control-Igat 1-1 ratio and 1-2 ratios. For cytokine rescue, soluble mIL-2, mll. 7,mILJ 5 and mIL-23 (all at 40 ng/ml) were added to the cell culture (FIG.14A), To examine the effects of costirnulacion, □CD28 (1 μg/ml) wasimmobilized together with □CD3 and 1g proteins at indicated ratios (FIG.14B). Cell proliferation was analyzed at 72 hr by examining CFSEdivision profiles. FIGS. 14C-D. To examine the suppressive activity ofPD-L3 OR VISTA in the presence of lower levels of costimulation,titrated amounts of ∘CD28 were coated together with anti-CD3 (2.Sμg/ml)and PD-L3 OR VISTA-1g fusion proteins or control-1g fusion protein (10to stimulate CD4+ T cell proliferation. Cell proliferation was analyzedat 72 hr, Percentages of proliferated CFSElow cells were quantified andshown in FIG. 14D. Duplicated wells were analyzed for all conditions,Representative CFSE profiles from three independent experiments areshown.

FIGS. 15A-D. PD-L3 OR VISTA expressed on antigen presenting cellssuppressed CD4 T cell proliferation. FIGS. 15A-C. The CHO cell line thatstably expresses MHCII molecule I-Ad and costimulation molecule B7-2 wasused as the parent cell line. Cells were transduced with retrovirusexpressing either PD-L3 OR VISTA-RFP or RFP control molecules.Transduced cells were sorted to achieve homogenous level of expression.To test their ability as antigen presenting cells, CHO-PD-L3 OR VISTA orCHO-RFP cells were mitomycin C treated and mixed with OVA-specifictransgenic CD4+ T cells D011.10, in the presence of titrated amount ofOVA peptide. Proliferation of DO 11 cells was analyzed at 72 hrs, eitherby CFSE division profiles (FIGS. 15A-B). or by tritium incorporation(FIG. 15C). D. bone marrow derived dendritic cells were transduced withRFP or B7B-H5-RFP retrovirus during 10-day culture period. TransducedCD11c+ RFP+ DCs and non-transduced CD 11c+ RFP− DCs were sorted and usedto stimulate OVA-specific transgenic CD4+ T cells OTII in the presenceof titrated amount of OVA peptide. Cell proliferation was analyzed onday 3 by examining CFSE division. For all experiments, duplicated wellswere analyzed for all conditions, and representative results from threeindependent experiments are shown.

FIG. 16. Surface expression level of PD-L3 OR VISTA in retrovirallytransduced bone marrow derived DCs. Bone marrow derived DCs (BMDC) werecultured in the presence of GM-CSF (20 ng/mml) and transduced witheither RFP or PD-L3 OR VISTA-RFP retrovinis as described in Methods. Onday 10, surface expression level of PD-L3 OR VISTA were analyzed oncultured BMDCs, and compared to freshly-isolated peritoneal macrophages.

FIG. 17 shows that anti-PDL3 mAb exhibits efficacy in a passive transferEAE model. In this adoptive transfer EAE model. donor SJL mice wereimmunized with CFA and PLP peptide. On day 10, total lymphocytes fromdraining LN were isolated, and cultured in vitro with PLP peptide,I1.,-23 (20 ng/ml) and anti-IFNg (10 μg/mail) for 4 days. Expanded CD4 Tcells were then purified and adoptively transferred into naive recipientmice. Disease progression was monitored and scored with: 0, no disease;0.5 loss of tail tone; 1: limp tad; 2: limp tail+ hind limb paresis;2.5: 1 hind limb paralysis; 3: both hind limb paralysis; 3.5: forelimbweakness: 4: hind limb paralysis+ unilateral forelimb paralysis. Micewere sacrificed when disease score reached 4. *, mice were sacrificed.

FIG. 18 shows than anti-PD-L3OR VISTA antibodies exhibit efficacy(reduce symptoms of arthritis) in a collagen-induced arthritis animalmodel.

FIG. 19 shows that VISTA expressed on antigen-presenting cellssuppressed CD4+ T cell proliferation. A monoclonal antibody clone 13F3neutralized VISTA mediated suppression in vitro. Total CD11bhi myeloidcells, or CD11bhiCD11c− monocytes and CD11bhiCD11c+ myeloid DCs weresorted from naïve splenocytes, irradiated and used to stimulate OTIItransgenic CD4+ T cells in the presence of OVA peptide. Cellproliferation was measured by tritium incorporation during the last 8hours of a 72-hour culture period. Triplicate wells were analyzed forall conditions.

FIG. 20 shows that an anti-VISTA antibody inhibited tumor growth in micetransplanted with MB49 tumor cells. Mice were inoculated with 2.4×10⁵MB49 tumor cells intradermally and tumor growth was monitored every 2-4days over a period of 3 weeks. Control-1g or a VISTA Mab (13F3) wereinjected every 2 days starting a week before tumor inoculationintraperitoneally. Each group had an n=8 mice. P-value.

FIGS. 21A-E show the antitumor effect of VISTA mabs in four differentmouse anti-tumor models.

FIG. 22 shows the potentiating effect of VISTA mabs on the efficacy of aCD40rrLR agonist vaccine.

FIG. 23 shows reduced VISTA expression on CNS myeloid cells. CNS cellswere isolated on day 9 and stained for CD4, CD11b and VISTA. Data istypical of at least 3 experiments.

FIG. 24 shows the impact of VISTA on the fate and function of T cells inan EAE model. VISTA-Ig suppresses CD4+ and CD8+ T cell proliferation.Purified CD8+ T cells were stimulated in vitro with αCD3+/− VISTA-Ig orcontrol Ig and CSFE dye dilution was measured. control Ig. See¹ fordetails.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Prior to describing the invention in more detail the followingdefinitions are provided.

As used herein, the term “immune cell” includes cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include lymphocytes, such as B cells and T cells; natural killercells; and myeloid cells, such as monocytes, macrophages, eosinophils,mast cells, basophils, and granulocytes.

As used herein, the term “T cell” includes CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells.

The term “antigen presenting cell” includes professional antigenpresenting cells (e.g., B lymphocytes, monocytes, dendritic cells, andLangerhans cells) as well as other antigen presenting cells (e.g.,keratinocytes, endothelial cells, astrocytes, fibroblasts, andoligodendrocytes).

The term ‘antigen” herein refers to antigen wherein the modulation ofthe immune response thereto may be therapeutically desired. In the caseof a desired enhanced immune response to particular antigens ofinterest, such antigens include, but are not limited to, infectiousdisease antigens for which a protective immune response may be elicitedare exemplary. For example, the antigens from HIV under considerationare the proteins gag, env, poi, tat, rev, nef, reverse transcriptase,and other HIV components. The E6 and E7 proteins from human papillomavirus are also under consideration. Furthermore, the EBNA1 antigen fromherpes simplex virus is also under consideration. Other viral antigensfor consideration are hepatitis viral antigens such as the S, M, and Lproteins of hepatitis B virus, the pre-S antigen of hepatitis B virus,and other hepatitis, e.g., hepatitis A, B, and C, viral components suchas hepatitis C viral RNA; influenza viral antigens such ashemagglutinin, neuraminidase, nucleoprotein, M2, and other influenzaviral components; measles viral antigens such as the measles virusfusion protein and other measles virus components; rubella viralantigens such as proteins E1 and E2 and other rubella virus components;rotaviral antigens such as VP7_(sc) and other rotaviral components;cytomegaloviral antigens such as envelope glycoprotein B and othercytomegaloviral antigen components; respiratory syncytial viral antigenssuch as the RSV fusion protein, the M2 protein and other respiratorysyncytial viral antigen components; herpes simplex viral antigens suchas immediate early proteins, glycoprotein D, and other herpes simplexviral antigen components; varicella zoster viral antigens such as gpI,gpII, and other varicella zoster viral antigen components; Japaneseencephalitis viral antigens such as proteins E, M-E, M-E-NS1, NS 1, NS1-NS2A, 80% E, and other Japanese encephalitis viral antigen components;rabies viral antigens such as rabies glycoprotein, rabies nucleoproteinand other rabies viral antigen components; West Nile virus prM and Eproteins; and Ebola envelope protein. See Fundamental Virology, SecondEdition, eds. Knipe, D. M. and, Howley P. M. (Lippincott Williams &Wilkins, New York, 2001) for additional examples of viral antigens. Inaddition, bacterial antigens are also disclosed. Bacterial antigenswhich can be used in the compositions and methods of the inventioninclude, but are not limited to, pertussis bacterial antigens such aspertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3,adenylate cyclase and other pertussis bacterial antigen components;diptheria bacterial antigens such as diptheria toxin or toxoid and otherdiptheria bacterial antigen components; tetanus bacterial antigens suchas tetanus toxin or toxoid and other tetanus bacterial antigencomponents; streptococcal bacterial antigens such as M proteins andother streptococcal bacterial antigen components; Staphylococcalbacterial antigens such as IsdA, IsdB, SdrD, and SdrE; gram-negativebacilli bacterial antigens such as lipopolysaccharides, flagellin, andother gram-negative bacterial antigen components; Mycobacteriumtuberculosis bacterial antigens such as mycolic acid, heat shock protein65 (HSP65), the 30 kDa major secreted protein, antigen 85A, ESAT-6, andother mycobacterial antigen components; Helicobacter pylori bacterialantigen components; pneumococcal bacterial antigens such as pneumolysin,pneumococcal capsular polysaccharides and other pneumococcal bacterialantigen components; haemophilus influenza bacterial antigens such ascapsular polysaccharides and other haemophilus influenza bacterialantigen components; anthrax bacterial antigens such as anthraxprotective antigen, anthrax lethal factor, and other anthrax bacterialantigen components; the F1 and V proteins from Yersinia pestis;rickettsiae bacterial antigens such as romps and other rickettsiaebacterial antigen components. Also included with the bacterial antigensdescribed herein are any other bacterial, mycobacterial, mycoplasmal,rickettsial, or chlamydial antigens. Examples of protozoa and otherparasitic antigens include, but are not limited to, Plasmodiumfalciparum antigens such as merozoite surface antigens, sporozoitesurface antigens, circumsporozoite antigens, gametocyte/gamete surfaceantigens, blood-stage antigen pf 1 55/RESA and other plasmodial antigencomponents; toxoplasma antigens such as SAG-1, p30 and other toxoplasmaantigen components; schistosomae antigens such asglutathione-S-transferase, paramyosin, and other schistosomal antigencomponents; Leishmania major and other leishmaniae antigens such asgp63, lipophosphoglycan and its associated protein and other leishmanialantigen components; and Trypanosoma cruzi antigens such as the 75-77 kDaantigen, the 56 kDa antigen and other trypanosomal antigen components.Examples of fungal antigens include, but are not limited to, antigensfrom Candida species, Aspergillus species, Blastomyces species,Histoplasma species, Coccidiodomycosis species, Malassezia furfur andother species, Exophiala werneckii and other species, Piedraia hortaiand other species, Trichosporum beigelii and other species, Microsporumspecies, Trichophyton species, Epidermophyton species, Sporothrixschenckii and other species, Fonsecaea pedrosoi and other species,Wangiella dermatitidis and other species, Pseudallescheria boydii andother species, Madurella grisea and other species, Rhizopus species,Absidia species, and Mucor species. Examples of prion disease antigensinclude PrP, beta-amyloid, and other prion-associated proteins.

In addition to the infectious and parasitic agents mentioned above,another area for desirable enhanced immunogenicity to a non-infectiousagent is in the area of dysproliferative diseases, including but notlimited to cancer, in which cells expressing cancer antigens aredesirably eliminated from the body. Tumor antigens which can be used inthe compositions and methods of the invention include, but are notlimited to, prostate specific antigen (PSA), breast, bladder, ovarian,testicular, melanoma, telomerase; multidrug resistance proteins such asP-glycoprotein; MAGE-1, alpha fetoprotein, carcinoembryonic antigen,mutant p53, papillomavirus antigens, gangliosides or othercarbohydrate-containing components of melanoma or other tumor cells. Itis contemplated by the invention that antigens from any type of tumorcell can be used in the compositions and methods described herein. Theantigen may be a cancer cell, or immunogenic materials isolated from acancer cell, such as membrane proteins. Included are survivin andtelomerase universal antigens and the MAGE family of cancer testisantigens. Antigens which have been shown to be involved in autoimmunityand could be used in the methods of the present invention to inducetolerance include, but are not limited to, myelin basic protein, myelinoligodendrocyte glycoprotein and proteolipid protein of multiplesclerosis and CII collagen protein of rheumatoid arthritis.

The antigen may be a portion of an infectious agent such as HZV-1, EBV,HBV, influenza virus, SARS virus, poxviruses, malaria, or HSV, by way ofnon-limiting examples, for which vaccines that mobilize strong T-cellmediated immunity (via dendritic cells) are needed.

The term “tumor” denotes at least one cell or cell mass in the form of atissue neoformation, in particular in the form of a spontaneous,autonomous and irreversible excess growth, which is more or lessdisinhibited, of endogenous tissue, which growth is as a rule associatedwith the more or less pronounced loss of specific cell and tissuefunctions. This cell or cell mass is not effectively inhibited, inregard to its growth, by itself or by the regulatory mechanisms of thehost organism, e.g. melanoma or carcinoma. Tumor antigens not onlyinclude antigens present in or on the malignant cells themselves, butalso include antigens present on the stromal supporting tissue of tumorsincluding endothelial cells and other blood vessel components.

As used herein, the term “immune response” includes T cell-mediatedand/or B cell-mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Bcell responses (e.g., antibody production) T cell responses (e.g.,cytokine production, and cellular cytotoxicity) and activation ofcytokine responsive cells, e.g., macrophages. As used herein, the term“downmodulation” with reference to the immune response includes adiminution in any one or more immune responses, while the term“upmodulation” with reference to the immune response includes anincrease in any one or more immune responses. It will be understood thatupmodulation of one type of immune response may lead to a correspondingdownmodulation in another type of immune response. For example,upmodulation of the production of certain cytokines (e.g., IL-10) canlead to downmodulation of cellular immune responses.

As used herein, the term “costimulatory receptor” includes receptorswhich transmit a costimulatory signal to an immune cell, e.g., CD28 orICOS. As used herein, the term “inhibitory receptors” includes receptorswhich transmit a negative signal to an immune cell

As used herein, the term “costimulate”, with reference to activatedimmune cells, includes the ability of a costimulatory molecule toprovide a second, non-activating, receptor-mediated signal (a“costimulatory signal”) that induces proliferation or effector function.For example, a costimulatory signal can result in cytokine secretion,e.g., in a T cell that has received a T cell-receptor-mediated signal.Immune cells that have received a cell receptor-mediated signal, e.g.,via an activating receptor, are referred to herein as “activated immunecells.”

An inhibitory signal as transduced by an inhibitory receptor can occureven if a costimulatory receptor (such as CD28 or ICOS) in not presenton the immune cell and, thus, is not simply a function of competitionbetween inhibitory receptors and costimulatory receptors for binding ofcostimulatory molecules (Fallarino et al. (1998) J. Exp. Med. 188:205).Transmission of an inhibitory signal to an immune cell can result inunresponsiveness, anergy or programmed cell death in the immune cell.Preferably, transmission of an inhibitory signal operates through amechanism that does not involve apoptosis.

As used herein the term “apoptosis” includes programmed cell death whichcan be characterized using techniques which are known in the art.Apoptotic cell death can be characterized, e.g., by cell shrinkage,membrane blebbing, and chromatin condensation culminating in cellfragmentation. Cells undergoing apoptosis also display a characteristicpattern of internucleosomal DNA cleavage.

The term “autoimmunity” or “autoimmune disease or condition” herein An“autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis such as acute arthritis, chronicrheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronicinflammatory arthritis, degenerative arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebralarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis,arthritis chronica progrediente, arthritis deformans, polyarthritischronica primaria, reactive arthritis, and ankylosing spondylitis),inflammatory hyperproliferative skin diseases, psoriasis such as plaquepsoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of thenails, dermatitis including contact dermatitis, chronic contactdermatitis, allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, colitis such as ulcerative colitis, colitisulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,necrotizing enterocolitis, and transmural colitis, and autoimmuneinflammatory bowel disease), pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, episcleritis), respiratory distresssyndrome, including adult or acute respiratory distress syndrome (ARDS),meningitis, inflammation of all or part of the uvea, iritis,choroiditis, an autoimmune hematological disorder, rheumatoidspondylitis, sudden hearing loss, IgE-mediated diseases such asanaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and/or brainstem encephalitis,uveitis, such as anterior uveitis, acute anterior uveitis, granulomatousuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterioruveitis, or autoimmune uveitis, glomerulonephritis (GN) with and withoutnephrotic syndrome such as chronic or acute glomerulonephritis such asprimary GN, immune-mediated GN, membranous GN (membranous nephropathy),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, allergic reaction, eczemaincluding allergic or atopic eczema, asthma such as asthma bronchiale,bronchial asthma, and auto-immune asthma, conditions involvinginfiltration of T cells and chronic inflammatory responses, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, systemic lupus erythematosus (SLE) or systemiclupus erythematodes such as cutaneous SLE, subacute cutaneous lupuserythematosus, neonatal lupus syndrome (NLE), lupus erythematosusdisseminatus, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I)diabetes mellitus, including pediatric insulin-dependent diabetesmellitus (IDDM), adult onset diabetes mellitus (Type II diabetes),autoimmune diabetes, idiopathic diabetes insipidus, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includinglymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis,vasculitides, including vasculitis (including large vessel vasculitis(including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), microscopic polyarteritis, CNS vasculitis,necrotizing, cutaneous, or hypersensitivity vasculitis, systemicnecrotizing vasculitis, and ANCA-associated vasculitis, such asChurg-Strauss vasculitis or syndrome (CSS)), temporal arteritis,aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia,Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiaperniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA),Factor VIII deficiency, hemophilia A, autoimmune neutropenia,pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNSinflammatory disorders, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet's orBehcet's disease, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, immune complexnephritis, antibody-mediated nephritis, neuromyelitis optica,polyneuropathies, chronic neuropathy such as IgM polyneuropathies orIgM-mediated neuropathy, thrombocytopenia (as developed by myocardialinfarction patients, for example), including thrombotic thrombocytopenicpurpura (TTP) and autoimmune or immune-mediated thrombocytopenia such asidiopathic thrombocytopenic purpura (ITP) including chronic or acuteITP, autoimmune disease of the testis and ovary including autoimmuneorchitis and oophoritis, primary hypothyroidism, hypoparathyroidism,autoimmune endocrine diseases including thyroiditis such as autoimmunethyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto'sthyroiditis); or subacute thyroiditis, autoimmune thyroid disease,idiopathic hypothyroidism, Grave's disease, polyglandular syndromes suchas autoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis,bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barresyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, primary biliary cirrhosis,pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AGED), autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal garnmopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal segmental glomerulosclerosis (FSGS),endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases,diabetic nephropathy, Dressler's syndrome, alopecia greata, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis,cystic fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis,Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection,echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirusinfection, rubella virus infection, post-vaccination syndromes,congenital rubella infection, Epstein-Barr virus infection, mumps,Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrineophthamopathy, chronic hypersensitivity pneumonitis,keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathicnephritic syndrome, minimal change nephropathy, benign familial andischemia-reperfusion injury, retinal autoimmunity, joint inflammation,bronchitis, chronic obstructive airway disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antobodies, non-malignant thymoma,vitiligo, SOD and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLesihmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoinunune disorders associated with collagendisease, rheumatism, neurological disease, ischemic re-perfusiondisorder, reduction in blood pressure response, vascular dysfunction,antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,reperfusion injury of myocardial or other tissues, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, acute serious inflammation, chronicintractable inflammation, pyelitis, pneumonocirrhosis, diabeticretinopathy, diabetic large-artery disorder, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; multiple myeloma and post-transplant lymphoproliferativedisorder (PTLD).

The phrase “allergic disease” refers to a disease involving allergicreactions. More specifically, an “allergic disease” is defined as adisease for which an allergen is identified, where there is a strongcorrelation between exposure to that allergen and the onset ofpathological change, and where that pathological change has been provento have an immunological mechanism. Herein, an immunological mechanismmeans that leukocytes show an immune response to allergen stimulation.Examples of allergens include mite antigens and pollen antigens.Representative allergic diseases include bronchial asthma, allergicrhinitis, atopic dermatitis, and pollen and insect allergies. Allergicdiathesis is a genetic factor that can be inherited by the children ofallergic parents. Familial allergic diseases are also called atopicdiseases, and the causative, genetically transmitted factor is atopicdiathesis. “Atopic dermatitis” is a general term for an atopic disease,especially diseases accompanied by dermatitis symptoms. Preferredexamples include allergic condition is selected from the groupconsisting of eczema, allergic rhinitis, hay fever, urticaria, and foodallergies. Allergic conditions include eczema, allergic rhinitis orcoryza, hay fever, bronchial asthma, urticaria (hives) and foodallergies, and other atopic conditions.

“Asthma”—refers to a disorder of the respiratory system characterized byinflammation, narrowing of the airways and increased reactivity of theairways to inhaled agents. Asthma is frequently, although notexclusively associated with atopic or allergic symptoms.

The phrase “inflammatory conditions or inflammatory disease” hereinincludes chronic or acute inflammatory diseases, including a disease orcondition selected from the group comprising: rheumatic diseases(including but not limited to rheumatoid arthritis, osteoarthritis,psoriatic arthritis) spondyloarthropathies (including but not limited toankylosing spondylitis, reactive arthritis, Reiter's syndrome), crystalarthropathies (including but not limited to gout, pseudogout, calciumpyrophosphate deposition disease), Lyme disease, polymyalgia rheumatica;connective tissue diseases (including but not limited to systemic lupuserythematosus, systemic sclerosis, polymyositis, dermatomyositis,Sjogren's syndrome); vasculitides (including but not limited topolyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome);inflammatory conditions including consequences of trauma or ischaemia,sarcoidosis; vascular diseases including atherosclerotic vasculardisease, atherosclerosis, and vascular occlusive disease (including butnot limited to atherosclerosis, ischaemic heart disease, myocardialinfarction, stroke, peripheral vascular disease), and vascular stentrestenosis; ocular diseases including uveitis, corneal disease, iritis,iridocyclitis, and cataracts;

The term cancer amenable for treatment by the present invention include,but not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemiaor lymphoid malignancies. More particular examples of such cancersinclude bladder, ovarian, melanoma, squamous cell cancer, lung cancer(including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.Preferably, the cancer is selected from the group consisting of breastcancer, colorectal cancer, rectal cancer, non-small cell lung cancer,non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, livercancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma. In an exemplary embodimenn (seeworking examples) the cancer is an early advanced (including metastatic)bladder, ovarian or melanoma. In another embodiment the cancer iscolorectal cancer. The cancerous conditions amenable for treatment ofthe invention include metastatic cancers wherein VISTA expression bymyeloid derived suppressor cells suppress antitumor responses andanti-invasive immune responses. The method of the present invention isparticularly suitable for the treatment of vascularized tumors.

The invention is also suitable for treating cancers in combination withchemotherapy or radiotherapy or other biologics and for enhancing theactivity thereof, i.e., in individuals wherein VISTA expression bymyeloid derived suppressor cells suppress antitumor responses and theefficacy of chemotherapy or radiotherapy or biologic efficacy. Anychemotherapeutic agent exhibiting anticancer activity can be usedaccording to the present invention. Preferably, the chemotherapeuticagent is selected from the group consisting of alkylating agents,antimetabolites, folic acid analogs, pyrimidine analogs, purine analogsand related inhibitors, vinca alkaloids, epipodopyyllotoxins,antibiotics, L-Asparaginase, topoisomerase inhibitor, interferons,platinum coordination complexes, anthracenedione substituted urea,methyl hydrazine derivatives, adrenocortical suppressant,adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens,antiandrogen, and gonadotropin-releasing hormone analog. Morepreferably, the chemotherapeutic agent is selected from the groupconsisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan,oxaliplatin, capecitabine, paclitaxel and doxetaxel. Two or morechemotherapeutic agents can be used in a cocktail to be administered incombination with administration of the anti-VEGF antibody. One preferredcombination chemotherapy is fluorouracil-based, comprising 5-FU and oneor more other chemotherapeutic agent(s). Suitable dosing regimens ofcombination chemotherapies are known in the art and described in, forexample, Saltz et al. (1999) Proc ASCO 18:233a and Douillard et al.(2000) Lancet 355:1041-7. The bilogic may be another immune potentiatorssuch as antibodies to PD-L1, PD-L2, CTLA-4 and PD-L1, PD-L2, CTLA-4fusion proteins as well as cytokines, growth factor antagonists andagonists, hormones and anti-cytokine antibodies.

Depending upon the form of the PD-L3 OR VISTA molecule that binds to areceptor, a signal can be either transmitted (e.g., by a multivalentform of a PD-L3 OR VISTA molecule that results in crosslinking of thereceptor or by a soluble form of PD-L3 OR VISTA that binds to Fcreceptors on antigen presenting cells) or inhibited (e.g., by a soluble,monovalent form of a PD-L3 OR VISTA molecule or a soluble form of PD-L3OR VISTA that is altered using methods known in the art such that itdoes not bind to Fc receptors on antigen presenting cells), e.g., bycompeting with activating forms of PD-*L3 OR VISTA molecules for bindingto the receptor. However, there are instances in which a solublemolecule can be stimulatory. The effects of the various modulatoryagents can be easily demonstrated using routine screening assays asdescribed herein.

As used herein, the term “activating receptor” includes immune cellreceptors that bind antigen, complexed antigen (e.g., in the context ofMHC molecules), or antibodies. Such activating receptors include T cellreceptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPSreceptors, complement receptors, and Fc receptors.

For example, T cell receptors are present on T cells and are associatedwith CD3 molecules. T cell receptors are stimulated by antigen in thecontext of MHC molecules (as well as by polyclonal T cell activatingreagents). T cell activation via the TCR results in numerous changes,e.g, protein phosphorylation, membrane lipid changes, ion fluxes, cyclicnucleotide alterations, RNA transcription changes, protein synthesischanges, and cell volume changes.

The term “B cell receptor” (BCR) as used herein includes the complexbetween membrane Ig (mIg) and other transmembrane polypeptides (e.g., Igalpha and Ig beta) found on B cells. The signal transduction function ofmIg is triggered by crosslinking of receptor molecules by oligomeric ormultimeric antigens. B cells can also be activated byanti-immunoglobulin antibodies. Upon BCR activation, numerous changesoccur in B cells, including tyrosine phosphorylation.

The term “Fc receptor” (FcRs) include cell surface receptors for the Fcportion of immunoglobulin molecules (Igs). Fc receptors are found onmany cells which participate in immune responses. Among the human FcRsthat have been identified so far, are those which recognize IgG(designated Fc gamma. R), IgE (Fc epsilon RI), IgA (Fc alpha R), andpolymerized IgM/A (Fc.mu. .alpha. R). FcRs are found in the followingcell types: Fc epsilon R I (mast cells), Fc epsilon. RH (manyleukocytes), Fc alpha. R (neutrophils), and Fc mu alpha. R (glandularepithelium, hepatocytes) (Hogg, N. (1988) Immunol. Today 9:185-86). Thewidely studied Fc gamma Rs are central in cellular immune defenses, andare responsible for stimulating the release of mediators of inflammationand hydrolytic enzymes involved in the pathogenesis of autoimmunedisease (Unkeless, J. C (1988) Annu. Rev. Immunol. 6:251-87). The FcgammaRs provide a crucial link between effector cells and thelymphocytes that secrete Ig, since the macrophage/monocyte,polymorphonuclear leukocyte, and natural killer (NK) cell Fc gamma Rsconfer an element of specific recognition mediated by IgG. Humanleukocytes have at least three different receptors for IgG: h Fc gamma.RI (found on monocytes/macrophages), hFc gamma RII (on monocytes,neutrophils, eosinophils, platelets, possibly B cells, and the K562 cellline), and Fc.gamma. III (on NK cells, neutrophils, eosinophils, andmacrophages).

With respect to T cells, transmission of a costimulatory signal to a Tcell involves a signaling pathway that is not inhibited by cyclosporinA. In addition, a costimulatory signal can induce cytokine secretion(e.g., IL-2 and/or IL-10) in a T cell and/or can prevent the inductionof unresponsiveness to antigen, the induction of anergy, or theinduction of cell death in the T cell.

As used herein, the term “inhibitory signal” refers to a signaltransmitted via an inhibitory receptor molecule on an immune cell. Sucha signal antagonizes a signal via an activating receptor (e.g., via aTCR, CD3, BCR, or Fc molecule) and can result, e.g., in inhibition of:second messenger generation; proliferation; or effector function in theimmune cell, e.g., reduced phagocytosis, antibody production, orcellular cytotoxicity, or the failure of the immune cell to producemediators (such as cytokines (e.g., IL-2) and/or mediators of allergicresponses); or the development of anergy.

As used herein, the term “unresponsiveness” includes refractivity ofimmune cells to stimulation, e.g., stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, e.g., because ofexposure to immunosuppressants or high doses of antigen.

As used herein, the term “anergy” or “tolerance” includes refractivityto activating receptor-mediated stimulation. Such refractivity isgenerally antigen-specific and persists after exposure to the tolerizingantigen has ceased. For example, anergy in T cells (as opposed tounresponsiveness) is characterized by lack of cytokine production, e.g.,IL-2. T cell anergy occurs when T cells are exposed to antigen andreceive a first signal (a T cell receptor or CD-3 mediated signal) inthe absence of a second signal (a costimulatory signal). Under theseconditions, reexposure of the cells to the same antigen (even ifreexposure occurs in the presence of a costimulatory molecule) resultsin failure to produce cytokines and, thus, failure to proliferate.Anergic T cells can, however, mount responses to unrelated antigens andcan proliferate if cultured with cytokines (e.g., IL-2). For example, Tcell anergy can also be observed by the lack of IL-2 production by Tlymphocytes as measured by ELISA or by a proliferation assay using anindicator cell line. Alternatively, a reporter gene construct can beused. For example, anergic T cells fail to initiate IL-2 genetranscription induced by a heterologous promoter under the control ofthe 5′ IL-2 gene enhancer or by a multimer of the API sequence that canbe found within the enhancer (Kang et al. (1992) Science 257:1134).

Modulation of a costimulatory signal results in modulation of effectorfunction of an immune cell. Thus, the term “PD-L3 OR VISTA activity”includes the ability of a PD-L3 OR VISTA polypeptide to bind its naturalbinding partner(s), the ability to modulate immune cell costimulatory orinhibitory signals, and the ability to modulate the immune response.

Modulation of an inhibitory signal in an immune cell results inmodulation of proliferation of and/or cytokine secretion by an immunecell.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, an “antisense” nucleic acid molecule comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid molecule can hydrogen bond to a sense nucleic acidmolecule.

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid molecule to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”or simply “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification., “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid molecule of the invention, such as a recombinantexpression vector of the invention, has been introduced. The terms “hostcell” and “recombinant host cell” are used interchangeably herein. Itshould be understood that such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

As used herein, a “transgenic animal” refers to a non-human animal,preferably a mammal, more preferably a mouse, in which one or more ofthe cells of the animal includes a “transgene”. The term “transgene”refers to exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, for example directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal.

As used herein, a “homologous recombinant animal” refers to a type oftransgenic non-human animal, preferably a mammal, more preferably amouse, in which an endogenous gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material and culturemedium when isolated from cells or produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thePD-L3 OR VISTA protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of PD-L3OR VISTA protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of PD-L3 OR VISTA proteinhaving less than about 30% (by dry weight) of non-PD-L3 OR VISTA protein(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-PD-L3 OR VISTA protein, still more preferablyless than about 10% of non-PD-L3 OR VISTA protein, and most preferablyless than about 5% non-PD-L3 OR VISTA protein. When the PD-L3 OR VISTAprotein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of PD-L3 OR VISTA protein in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of PD-L3 OR VISTA protein having less than about30% (by dry weight) of chemical precursors or non-PD-L3 OR VISTAchemicals, more preferably less than about 20% chemical precursors ornon-PD-L3 OR VISTA chemicals, still more preferably less than about 10%chemical precursors or non-PD-L3 OR VISTA chemicals, and most preferablyless than about 5% chemical precursors or non-PD-L3 OR VISTA chemicals.

The term “antibody”, as used herein, includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”), as well as wholeantibody molecules. The term “antigen-binding portion”, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g, PD-L3 OR VISTA). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody; (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VIIregions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al.1998 Nat. Biotechnol. 16:778). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Any VH and VL sequences of specific scFv can be linked tohuman immunoglobulin constant region cDNA or genomic sequences, in orderto generate expression vectors encoding complete IgG molecules or otherisotypes. VH and Vl can also be used in the generation of Fab, Fv, orother fragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P. et al. (1993) ProcNatl. Acad. Sci. USA 90:6444-6448; Poljak, R. J. et al. (1994) Structure2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M. et al. (1995) Hum.Antibodies Hybridomas 6:93-101) and use of a cysteine residue, a markerpeptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M. et al. (1994) MolImmunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)2fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof, e.g., humanized, chimeric, etcPreferably, antibodies of the invention bind specifically orsubstantially specifically to PD-L3 OR VISTA molecules. The terms“monoclonal antibodies” and “monoclonal antibody composition”, as usedherein, refer to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of an antigen, whereas the term “polyclonalantibodies” and “polyclonal antibody composition” refer to a populationof antibody molecules that contain multiple species of antigen bindingsites capable of interacting with a particular antigen. A monoclonalantibody composition, typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

The term “humanized antibody”, as used herein, is intended to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs. The term “humanized antibody”, as used herein, also includesantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds PD-L3 OR VISTA is substantially free of antibodies thatspecifically bind antigens other than PD-L3 OR VISTA). Moreover, anisolated antibody may be substantially free of other cellular materialand/or chemicals.

An “oligomerization domain” herein refers to a domain that when attachedto a VISTA extracellular domain or fragment thereof, facilitatesoligomerization. Said oligomerization domains comprise self-associating.alpha.-helices, for example, leucine zippers, that can be furtherstabilized by additional disulfide bonds. The domains are designed to becompatible with vectorial folding across a membrane, a process thoughtto facilitate in vivo folding of the polypeptide into a functionalbinding protein. Examples thereof are known in the art and include byway of example coiled GCN4 and COMP.

The .alpha.-helical coiled coil is probably the most widespread subunitoligomerization motif found in proteins. Accordingly, coiled coilsfulfill a variety of different functions. In several families oftranscriptional activators, for example, short leucine zippers play animportant role in positioning the DNA-binding regions on the DNA(Ellenberger et al., 1992, Cell 71:1223-1237). Coiled coils are alsoused to form oligomers of intermediate filament proteins. Coiled-coilproteins furthermore appear to play an important role in both vesicleand viral membrane fusion (Skehel and Wiley, 1998, Cell 95:871-874). Inboth cases hydrophobic sequences, embedded in the membranes to be fused,are located at the same end of the rod-shaped complex composed of abundle of long .alpha.-helices. This molecular arrangement is believedto cause close membrane apposition as the complexes are assembled formembrane fusion. The coiled coil is often used to controloligomerization. It is found in many types of proteins, includingtranscription factors such as, but not limited to GCN4, viral fusionpeptides, SNARE complexes and certain tRNA synthetases, among others.Very long coiled coils are found in proteins such as tropomyosin,intermediate filaments and spindle-pole-body components. Coiled coilsinvolve a number of .alpha.-helices that are supercoiled around eachother in a highly organized manner that associate in a parallel or anantiparallel orientation. Although dimers and trimers are the mostcommon. The helices may be from the same or from different proteins. Thecoiled-coil is formed by component helices coming together to bury theirhydrophobic seams. As the hydrophobic seams twist around each helix, sothe helices also twist to coil around each other, burying thehydrophobic seams and forming a supercoil. It is the characteristicinterdigitation of side chains between neighbouring helices, known asknobs-into-holes packing, that defines the structure as a coiled coil.The helices do not have to run in the same direction for this type ofinteraction to occur, although parallel conformation is more common.Antiparallel conformation is very rare in trimers and unknown inpentamers, but more common in intramolecular dimers, where the twohelices are often connected by a short loop. In the extracellular space,the heterotrimeric coiled-coil protein laminin plays an important rolein the formation of basement membranes. Other examples are thethrombospondins and cartilage oligomeric matrix protein (COMP) in whichthree (thrombospondins 1 and 2) or five (thrombospondins 3, 4 and COMP)chains are connected. The molecules have a flower bouquet-likeappearance, and the reason for their oligomeric structure is probablythe multivalent interaction of the C-terminal domains with cellularreceptors. The yeast transcriptional activator GCN4 is 1 of over 30identified eukaryotic proteins containing the basic region leucinezipper (bZIP) DNA-binding motif (Ellenberger et al., 1992, Cell71:1223-1237). The bZIP dimer is a pair of continuous alpha helices thatform a parallel coiled-coil over their carboxy-terminal 34 residues andgradually diverge toward their amino termini to pass through the majorgroove of the DNA binding site. The coiled-coil dimerization interfaceis oriented almost perpendicular to the DNA axis, giving the complex theappearance of the letter T. bZIP contains a 4-3 heptad repeat ofhydrophobic and nonpolar residues that pack together in a parallelalpha-helical coiled-coil (Ellenberger et al., 1992, Cell 71:1223-1237).The stability of the dimer results from the side-by-side packing ofleucines and nonpolar residues in positions a and d of the heptadrepeat, as well as a limited number of intra- and interhelical saltbridges, shown in a crystal structure of the GCN4 leucine zipper peptide(Ellenberger et al., 1992, Cell 71:1223-1237). Another example is CMP(matrilin-1) isolated from bovine tracheal cartilage as a homotrimer ofsubunits of Mr 52,000 (Paulsson and Heinegard, 1981, Biochem J.197:367-375), where each subunit consists of a vWFA1 module, a singleEGF domain, a vWFA2 module and a coiled coil domain spanning fiveheptads (Kiss et al., 1989, J. Biol. Chem. 264:8126-8134; Hauser andPaulsson, 1994, J. Biol. Chem. 269:25747-25753). Electron microscopy ofpurified CMP showed a bouquet-like trimer structure in which eachsubunit forms an ellipsoid emerging from a common point corresponding tothe coiled coil (Hauser and Paulsson, 1994, J. Biol. Chem.269:25747-25753). The coiled coil domain in matrilin-1 has beenextensively studied. The trimeric structure is retained after completereduction of interchain disulfide bonds under non-denaturing conditions(Hauser and Paulsson, 1994, J. Biol. Chem. 269:25747-25753). Yet anotherexample is Cartilage Oligomeric Matrix Protein (COMP). A non-collagenousglycoprotein, COMP, was first identified in cartilage (Hedbom et al.,1992, J. Biol. Chem. 267:6132-6136). The protein is a 524 kDahomopentamer of five subunits which consists of an N-terminal heptadrepeat region (cc) followed by four epidermal growth factor (EGF)-likedomains (EF), seven calcium-binding domains (T3) and a C-terminalglobular domain (TC). According to this domain organization, COMPbelongs to the family of thrombospondins. Heptad repeats (abcdefg).sub.nwith preferentially hydrophobic residues at positions a and dform-helical coiled-coil domains (Cohen and Parry, 1994, Science263:488-489). Recently, the recombinant five-stranded coiled-coil domainof COMP (COMPcc) was crystallized and its structure was solved at 0.2run resolution (Malashkevich et al., 1996, Science 274:761-765).

The term “family” when referring to the polypeptide and nucleic acidmolecules of the invention is intended to mean two or more polypeptideor nucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first polypeptide of human origin, as well asother, distinct polypeptides of human origin or alternatively, cancontain homologues of non-human origin, e.g., monkey polypeptides.Members of a family may also have common functional characteristics.

For example, the family of PD-L3 OR VISTA polypeptides of the presentinvention preferably comprises least one “signal peptide domain”. Asused herein, a “signal sequence” or “signal peptide” includes a peptidecontaining about 15 or more amino acids which occurs at the N-terminusof secretory and membrane bound polypeptides and which contains a largenumber of hydrophobic amino acid residues. For example, a signalsequence contains at least about 10-30 amino acid residues, preferablyabout 15-25 amino acid residues, more preferably about 18-20 amino acidresidues, and even more preferably about 19 amino acid residues, and hasat least about 35-65%, preferably about 38-50%, and more preferablyabout 40-45% hydrophobic amino acid residues (e.g., Valine, Leucine.Isoleucine or Phenylalanine). Such a “signal sequence”, also referred toin the art as a “signal peptide”, serves to direct a polypeptidecontaining such a sequence to a lipid bilayer, and is cleaved insecreted and membrane bound polypeptides. As described infra a signalsequence was identified in the amino acid sequence of native human PD-L3OR VISTA and was also identified in the amino acid sequence of nativemouse PD-L3 OR VISTA

In another embodiment of the invention, a PD-L3 OR VISTA polypeptide ofthe present invention is identified based on the presence of a“transmembrane domain”. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15 amino acid residues inlength which spans the plasma membrane. More preferably, a transmembranedomain includes about at least 20, 25, 30, 35, 40, or 45 amino acidresidues and spans the plasma membrane. Transmembrane domains are richin hydrophobic residues, and typically have an alpha-helical structure.In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or moreof the amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domainsare described in, for example, Zagotta, W. N. et al. (1996) Annu. Rev.Neurosci. 19:235-263, the contents of which are incorporated herein byreference. The transmembrane domain region of PDL3 are identified herein(see e.g., FIG. 1).

In another embodiment, a PD-L3 OR VISTA molecule of the presentinvention is identified based on the absence of an “IgC domain” and thepresence of an “IgV domain” in the polypeptide or corresponding nucleicacid molecule. As used herein, IgV and IgC domains are recognized in theart as Ig superfamily member domains. These domains correspond tostructural units that have distinct folding patterns called Ig folds. Igfolds are comprised of a sandwich of two beta sheets, each consisting ofantiparallel beta strands of 5-10 amino acids with a conserved disulfidebond between the two sheets in most, but not all, domains. IgC domainsof Ig, TCR, and MHC molecules share the same types of sequence patternsand are called the C1 set within the Ig superfamily. Other IgC domainsfall within other sets. IgV domains also share sequence patterns and arecalled V set domains. IgV domains are longer than C-domains and form anadditional pair of beta strands. The amino acid residues of the nativehuman and murine PD-L3 OR VISTA polypeptide, constituting the IgV domsincan be seen in FIG. 1. The presence of an IgV domain is likely requiredfor binding of PD-L3 OR VISTA to its natural binding partner(s)

In another embodiment, a PD-L3 OR VISTA molecule of the presentinvention is identified based on the presence of a “extracellulardomain” in the polypeptide or corresponding nucleic acid molecule. Asused herein, the term “extracellular domain” represents the N-terminalamino acids which extend as a tail from the surface of a cell. Anextracellular domain of the present invention includes an IgV domain andmay include a signal peptide domain. (See FIG. 1).

In still another embodiment, a PD-L3 OR VISTA molecule of the presentinvention is identified based on the presence of a “cytoplasmic domain”in the polypeptide or corresponding nucleic acid molecule. As usedherein, the term “cytoplasmic domain” represents the C-terminal aminoacids which extend as a tail into the cytoplasm of a cell. predicted tocomprise cytoplasmic domains.

In a preferred embodiment, the PD-L3 OR VISTA molecules of the inventioninclude at least one or more of the following domains: a signal peptidedomain, an IgV domain, an extracellular domain, a transmembrane domain,and a cytoplasmic domain.

Isolated polypeptides of the present invention, preferably PD-L3 ORVISTA polypeptides, have an amino acid sequence sufficiently identicalto the amino acid sequence of SEQ ID NO: 2 or 4, or 5 or are encoded bya nucleotide sequence sufficiently identical to SEQ ID NO: 1 or 3 orfragment or complement thereof. As used herein, the term “sufficientlyidentical” refers to a first amino acid or nucleotide sequence whichcontains a sufficient or minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences sharecommon structural domains or motifs and/or a common functional activity.For example, amino acid or nucleotide sequences which share commonstructural domains have at least 30%, 40%, or 50% homology, preferably60% homology, more preferably 70%-80%, and even more preferably 90-95%homology across the amino acid sequences of the domains and contain atleast one and preferably two structural domains or motifs, are definedherein as sufficiently identical. Furthermore, amino acid or nucleotidesequences which share at least 30%, 40%, or 50%, preferably 60%, morepreferably 70-80%, or 90-95% homology and share a common functionalactivity are defined herein as sufficiently identical.

As used interchangeably herein, “PD-L3 OR VISTA activity”, “biologicalactivity of PD-L3 OR VISTA” or “functional activity of PD-L3 OR VISTA”,refers to an activity exerted by a PD-L3 OR VISTA protein, polypeptideor nucleic acid molecule on a PD-L3 OR VISTA-responsive cell or tissue,or on a PD-L3 OR VISTA polypeptide binding partner, as determined invivo, or in vitro, according to standard techniques. These activitiesinclude modulating CD4+ and CD8+ T cell proliferation and cytokineproduction. In another embodiment, a PD-L3 OR VISTA activity is a directactivity, such as an association with a PD-L3 OR VISTA binding partner.As used herein, a “target molecule” or “binding partner” is a moleculewith which a PD-L3 OR VISTA polypeptide binds or interacts in nature,i.e., expressed on a T cell, such that PD-L3 OR VISTA-mediated functionis achieved. Alternatively, a PD-L3 OR VISTA activity is an indirectactivity, such as a cellular signaling activity mediated by the PD-L3 ORVISTA polypeptide. The biological activities of PD-L3 OR VISTA aredescribed herein. For example, the PD-L3 OR VISTA polypeptides and PD-L3OR VISTA agonists or antagonists of the present invention can have oneor more of the following activities: (1) suppresses or promotes CD4+ andCD8+ T cell proliferation, (2) suppresses or promotes cytokineproduction (3) functions as a regulatory ligand that negativelyregulates T cell responses during cognate interactions between T cellsand myeloid derived APCs (4) negatively regulates CD4+ T cell responsesby suppressing early TCR activation and arresting cell division, butwith minimum direct impact on apoptosis, (5) suppresses or promotesantigen-specific T cell activation during cognate interactions betweenAPCs and T cells and/or (6) suppresses or promotes T cell-mediatedimmune responses; (7) modulate activation of immune cells, e.g., Tlymphocytes, and (8) modulate the immune response, e.g., inflammatoryimmune response of an organism, e.g., a mouse or human organism.

Accordingly, another embodiment of the invention features isolated PD-L3OR VISTA proteins and polypeptides that modulate one or more PD-L3 ORVISTA activities. These polypeptides will include PD-L3 OR VISTApolypeptides having one or more of the following domains: a signalpeptide domain, an IgV domain, an extracellular domain, a transmembranedomain, and a cytoplasmic domain, and, preferably, a PD-L3 OR VISTAactivity.

Additional preferred PD-L3 OR VISTA polypeptides may have at least oneextracellular domain, and one or more of a signal peptide domain, an 1₅Vdomain, an transmembrane domain, and a cytoplasmic domain, and are,preferably, encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule comprising a complement of the nucleotide sequenceof SEQ ID NO: 1 or 3 herein. The nucleotide and amino acid sequencessequence of the exemplified isolated human and marine PD-L3 OR VISTAcDNA and the predicted amino acid sequence of the human PD-L3 OR VISTApolypeptide are contained in the sequence listing herein.

Human VISTA or PD-L3 OR VISTA was identified as an upregulated moleculein a T cell transcriptional profiling screen. Our characterization of anidentical 930 bp gene product recovered from a murine CD4⁺ T-cell cDNAlibrary confirmed the size and sequence. Silico-sequence and structuralanalysis predicts a type I transmembrane protein of 309 amino acids uponmaturation. Its extracellular domain contains a single extracellularIg-V domain of 136 amino acids, which is linked to a 23-amino acid stalkregion, a 21-residue transmembrane segment, and a 97-amino acidcytoplasmic domain. The cytoplasmic tail of VISTA does not contain anysignaling domains. A BLAST sequence search with the VISTA Ig-V domainidentified PD-L1 of the B7 family as the closest evolutionarily relatedprotein with a borderline significant e-value score. A structure basedsequence alignment of VISTA with the B7 family members PD-L1, PD-L2,B7-H3, and B7-H4 highlights several amino acids that are systematicallyconserved in all Ig-V domain proteins.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. PD-L3 OR VISTA Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode PD-L3 OR VISTA polypeptides or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identify PD-L3 OR VISTA-encoding nucleic acidmolecules (e.g., PD-L3 OR VISTA mRNA) and fragments for use as PCRprimers for the amplification or mutation of PD-L3 OR VISTA nucleic acidmolecules. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acidmolecule is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and 3′ ends of the nucleic acidmolecule) in the genomic DNA of the organism from which the nucleic acidis derived. For example, in various embodiments, the isolated PD-L3 ORVISTA nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid molecule is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium, when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 1,3, or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. Using all or portion of thenucleic acid sequence of SEQ ID NO: 1, or 3 as a hybridization probe,PD-L3 OR VISTA nucleic acid molecules can be isolated using standardhybridization and cloning techniques (e.g., as described in Sambrook, J.et al. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO: 1, 3, or an ortholog or variant can be isolated by the polymerasechain reaction (PCR) using synthetic oligonucleotide primers designedbased upon the sequence of SEQ ID NO: 1, 2, 3, 4 or 5.

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or, alternatively, genomic DNA as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to PD-L3 nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In a preferred embodiment, an isolated PD-L3 OR VISTA encoding nucleicacid molecule of the invention comprises the nucleotide sequence shownin SEQ ID NO: 1, or 3, or a fragment thereof In another embodiment thenucleic acid molecule of the invention comprises a nucleic acid moleculewhich is a complement of the nucleotide sequence shown in SEQ ID NO: 1,or 3, or a portion of any of these nucleotide sequences. A nucleic acidmolecule which is complementary to the nucleotide sequence shown in SEQID NO: 1, or 3, is one which is sufficiently complementary to thenucleotide sequence shown in SEQ ID NO: 1, or 3 such that it canhybridize to the nucleotide sequence shown in SEQ ID NO: 1, or 3respectively, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 70%, 75%, 80%, 85%, 90%, 0.91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the entire length of the nucleotidesequence shown in SEQ ID NO: 1 or 3, or a portion of any of thesenucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO: 1, or 3, for example,a fragment which can be used as a probe or primer or a fragment whichencodes a portion of a PD-L3 OR VISTA polypeptide, e.g., a biologicallyactive portion of a PD-L3 OR VISTA-polypeptide. The nucleotide sequencesdetermined from the cloning of the human PD-L2 gene allow for thegeneration of probes and primers designed for use in identifying and/orcloning other PD-L2 family members, as well as PD-L3 OR VISTA homologuesfrom other species. The probe/primer typically comprises substantiallypurified oligonucleotide. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12 or 15, preferably about 20 or 25, more preferablyabout 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of asense sequence of SEQ ID NO: 1, or 3; of an anti-sense sequence of SEQID NO: 1, 3, or a naturally occurring allelic variant or mutant of SEQID NO: 1, or 3.

In one embodiment, a nucleic acid molecule of the present inventioncomprises a nucleotide sequence which is greater than about 50-100,100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900,900-950 or more nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of SEQ ID NO: 1, or3, or the complement thereof. In a further embodiment, a nucleic acidmolecule of the present invention comprises a nucleotide sequence whichis greater than about 880-900, 900-950, 950-1000, 1000-1050, 1050-1100,1100-1150 or more nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of SEQ ID NO: 1 or3, or the complement thereof. In yet another embodiment, a nucleic acidmolecule of the present invention comprises a nucleotide sequence whichis greater than 50-100, 100-150, 150-200, 200-250, 250-300 or morenucleotides in length and hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the coding region inSEQ ID NO: 1 or 3, or a complement thereof. In yet a further embodiment,a nucleic acid molecule of the present invention comprises a nucleotidesequence which is greater than about 50-100, 100-150, 150-200, 200-250,250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650,650-700, 700-750, 750-800, 850-900, 900-950, or more nucleotides inlength, includes at least about 15 (i.e., 15 contiguous) nucleotides ofthe sequence comprising the coding region of SEQ ID NO: 1 or 3, or acomplement thereof, and hybridizes under stringent conditions to anucleic acid molecule comprising the nucleotide sequence shown in SEQ IDNO: 1, or 3 a complement thereof.

Probes based on the PD-L3 OR VISTA nucleotide sequences can be used todetect transcripts or genomic sequences encoding the same or homologouspolypeptides. In preferred embodiments, the probe further comprises alabel group attached thereto, e.g., the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress a PD-L3 OR VISTApolypeptide, such as by measuring a level of a PD-L3 OR VISTA-encodingnucleic acid in a sample of cells from a subject e.g., detecting PD-L3OR VISTA mRNA levels or determining whether a genomic PD-L3 OR VISTAgene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aPD-L3 OR VISTA polypeptide” can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO: 1, or 3 which encodes apolypeptide having a PD-L3 OR VISTA biological activity (e.g., theability to bind to its natural binding partner(s) and/or modulate immunecell activity), expressing the encoded portion of the PD-L3 OR VISTApolypeptide (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of the PD-L3 OR VISTA polypeptide.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO: 1, or 3 due todegeneracy of the genetic code and thus encode the same PD-L3 OR VISTApolypeptides as those encoded by the nucleotide sequence shown in SEQ IDNO: 1, or 3. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a polypeptide having anamino acid sequence shown in SEQ ID NO: 2, 4 or 5.

In addition to the PD-L3 OR VISTA nucleotide sequences shown in SEQ IDNO: 1, and 3, it will be appreciated by those skilled in the art thatDNA sequence polymorphisms that lead to changes in the amino acidsequences of the PD-L3 OR VISTA polypeptides may exist within apopulation (e.g., the human population). Such genetic polymorphism inthe PD-L3 OR VISTA genes may exist among individuals within a populationdue to natural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include an openreading frame encoding a PD-L3 OR VISTA polypeptide, preferably amammalian PD-L3 OR VISTA polypeptide, and can further include non-codingregulatory sequences, and introns.

Allelic variants of human or mouse PD-L3 OR VISTA include bothfunctional and non-functional PD-L3 OR VISTA polypeptides. Functionalallelic variants are naturally occurring amino acid sequence variants ofthe human or mouse PD-L3 OR VISTA polypeptide that maintain the abilityto bind natural PD-L3 OR VISTA binding partner(s) and/or modulate CD4+and CD8+ T cell proliferation and cytokine production and lymphocyteactivation. Functional allelic variants will typically contain onlyconservative substitution of one or more amino acids of SEQ ID NO: 2, 4or 5, or substitution, deletion or insertion of non-critical residues innon-critical regions of the polypeptide.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human or mouse PD-L3 OR VISTA polypeptide thatdo not have the ability to either bind natural PD-L3 OR VISTA bindingpartners, and/or modulate any of the PD-L3 OR VISTA activities describedherein. Non-functional allelic variants will typically contain anon-conservative substitution, deletion, or insertion or prematuretruncation of the amino acid sequence of SEQ ID NO: 2, 4 or 5, or asubstitution, insertion or deletion in critical residues or criticalregions of the polypeptide, e.g., in an IgV domain.

The present invention further provides non-human, non-mouse orthologs ofthe human or mouse PD-L3 OR VISTA polypeptide. Orthologs of the human ormouse PD-L3 OR VISTA polypeptide are polypeptides that are isolated fromnon-human, non-mouse organisms and possess the same binding activityand/or lymphocyte activation-modulating activity, and ability tomodulate CD4+ and CD8+ T cell proliferation and cytokine production asthe human and murine PD-L3 OR VISTA polypeptides disclosed herein.Orthologs of the human or mouse PD-L3 polypeptide can readily beidentified as comprising an amino acid sequence that is substantiallyidentical to SEQ ID NO: 2, 4 or 5.

Moreover, nucleic acid molecules encoding other PD-L3 OR VISTA familymembers and, thus, which have a nucleotide sequence which differs fromthe PD-L3 OR VISTA sequences of SEQ ID NO: 1, or 3 are intended to bewithin the scope of the invention. For example, another PD-L3 OR VISTAcDNA can be identified based on the nucleotide sequence of mouse orhuman PD-L3 OR VISTA. Moreover, nucleic acid molecules encoding PD-L3 ORVISTA polypeptides from different species, and which, thus, have anucleotide sequence which differs from the PD-L3 OR VISTA sequences ofSEQ ID NO: 1, or 3 are intended to be within the scope of the invention.For example, a monkey PD-L3 OR VISTA cDNA can be identified based on thenucleotide sequence of the mouse or human PD-L3 OR VISTA.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the PD-L3 OR VISTA cDNAs of the invention can be isolatedbased on their homology to the PD-L2 nucleic acids disclosed hereinusing the cDNAs disclosed herein, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. Nucleic acid molecules correspondingto natural allelic variants and homologues of the PD-L3 OR VISTA cDNAsof the invention can further be isolated by mapping to the samechromosome or locus as the PD-L3 OR VISTA gene.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the coding region of the nucleotide sequence of SEQ ID NO: 1or 3. In other embodiment, the nucleic acid is at least 700, 750, 800,850, 880-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150 or morenucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc (1995), sections 2, 4 and 6. Additional stringentconditions can be found in Molecular Cloning: A Laboratory Manual,Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989), chapters 7, 9 and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4 times or6 times sodium chloride/sodium citrate (SSC), at about 65-70 degrees C.(or hybridization in 4 times SSC plus 50% formamide at about 42-50degrees C.) followed by one or more washes in 1×SSC, at about 65-70degrees C. A further preferred, non-limiting example of stringenthybridization conditions includes hybridization at 6 times SSC at 45degrees C., followed by one or more washes in 0.2 times SSC, 0.1% SDS at65 degrees C. A preferred, non-limiting example of highly stringenthybridization conditions includes hybridization in 1 times SSC, at about65-70 degrees C. (or hybridization in 1 times SSC plus 50% formamide atabout 42-50 degrees C.) followed by one or more washes in 0.3 times SSC,at about 65-70 degrees C. A preferred, non-limiting example of reducedstringency hybridization conditions includes hybridization in 4 times or6 times SSC, at about 50-60 degrees C. (or alternatively hybridizationin 6 times SSC plus 50% formamide at about 40-45 degrees C.) followed byone or more washes in 2 times SSC, at about 50-60 degrees C. Rangesintermediate to the above-recited values, e.g., at 65-70 degrees C. orat 42-50 degrees C. are also intended to be encompassed by the presentinvention. SSPE (1 times SSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mMEDTA, pH 7.4) can be substituted for SSC (1 times SSC is 0.15M NaCl and15 mM sodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes each after hybridization is complete. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10 degrees C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(degrees C.)-2 (# of A+T bases)+4(# of G+C bases). For hybrids between18 and 49 base pairs in length, Tm (degrees C.)=81.5+16.6(log10[Na+])+0.41(% G+C)−(600/N), where N is the number of bases in thehybrid, and [Na+1] is the concentration of sodium ions in thehybridization buffer ([Na.+] for 1 times SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH2PO4, 7% SDS at about 65 degrees C.,followed by one or more washes at 0.02M NaH2PO4, 1% SDS at 65 degreesC., see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA81:1991-1995 (or alternatively 0.2 times SSC, 1% SDS).

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO: 1,or 3 or corresponds to a naturally-occurring nucleic acid molecule. Asused herein, a “naturally-occurring” nucleic acid molecule refers to anRNA or DNA molecule having a nucleotide sequence that occurs in nature(i.e., encodes a natural polypeptide).

In addition to naturally-occurring allelic variants of the PD-L3 ORVISTA sequences that may exist in the population, the skilled artisanwill further appreciate that changes can be introduced by mutation intothe nucleotide sequences of SEQ ID NO: 1 or 3, thereby leading tochanges in the amino acid sequence of the encoded PD-L3 OR VISTApolypeptides, without altering the functional ability of the PD-L3 ORVISTA polypeptides. For example, nucleotide substitutions leading toamino acid substitutions at “non-essential” amino acid residues can bemade in the sequence of SEQ ID NO: 1, or 3. A “non-essential” amino acidresidue is a residue that can be altered from the wild-type sequence ofPD-L3 OR VISTA (e.g., the sequence of SEQ ID NO: 2, 4 or 5) withoutaltering the biological activity, whereas an “essential” amino acidresidue is required for biological activity. For example, amino acidresidues that are conserved among the PD-L3 OR VISTA polypeptides of thepresent invention, e.g., those present in an extracellular domain, arepredicted to be particularly unamenable to alteration. Furthermore,additional amino acid residues that are conserved between the PD-L3 ORVISTA polypeptides of the present invention and other members of thePD-L3 OR VISTA family are not likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding PD-L3 OR VISTA polypeptides that contain changes inamino acid residues that are not essential for activity. Such PD-L3 ORVISTA polypeptides differ in amino acid sequence from SEQ ID NO: 2, 4 or5, yet retain biological activity. In one embodiment, the isolatednucleic acid molecule comprises a nucleotide sequence encoding apolypeptide, wherein the polypeptide comprises an amino acid sequence atleast about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to SEQ ID NO: 2, 4 or 5.

An isolated nucleic acid molecule encoding a PD-L3 or VISTA polypeptideidentical to the polypeptide of SEQ ID NO: 2, 4 or 5 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO: 1 or 3 such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded polypeptide. Mutations can be introduced into SEQ ID NO: 1 or 3by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g. lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a PD-L3OR VISTA polypeptide is preferably replaced with another amino acidresidue from the same side chain family. Alternatively, in anotherembodiment, mutations can be introduced randomly along all or part of aPD-L3 OR VISTA coding sequence, such as by saturation mutagenesis, andthe resultant mutants can be screened for PD-L3 OR VISTA biologicalactivity to identify mutants that retain activity. Following mutagenesisof SEQ ID NO: 1, or 3, the encoded polypeptide can be expressedrecombinantly and the activity of the polypeptide can be determined.

In a preferred embodiment, a mutant PD-L3 OR VISTA polypeptide can beassayed for the ability to bind to and/or modulate the activity of anatural PD-L3 OR VISTA binding partner, to modulate intra- orintercellular signaling, modulate activation of T lymphocytes, and/ormodulate the immune response of an organism.

Yet another aspect of the invention pertains to isolated nucleic acidmolecules encoding a PD-L3 OR VISTAPD-L3 OR VISTA OR VISTA fusionproteins. Such nucleic acid molecules, comprising at least a firstnucleotide sequence encoding a PD-L3 OR VISTAPD-L3 OR VISTA OR VISTAprotein, polypeptide or peptide operatively linked to a secondnucleotide sequence encoding a non-PD-L3 OR VISTA protein, polypeptideor peptide, can be prepared by standard recombinant DNA techniques.

In addition to the nucleic acid molecules encoding PD-L3 OR VISTApolypeptides described above, another aspect of the invention pertainsto isolated nucleic acid molecules which are antisense thereto. An“antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a polypeptide, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire PD-L3 OR VISTA coding strand, orto only a portion thereof. In one embodiment, an antisense nucleic acidmolecule is antisense to a “coding region” of the coding strand of anucleotide sequence encoding a PD-L3 OR VISTA. The term “coding region”refers to the region of the nucleotide sequence comprising codons whichare translated into amino acid residues. In another embodiment, theantisense nucleic acid molecule is antisense to a “noncoding region” ofthe coding strand of a nucleotide sequence encoding PD-L. The term“noncoding region” refers to 5′ and 3′ sequences which flank the codingregion that are not translated into amino acids (also referred to as 5′and 3′ untranslated regions). Given the coding strand sequences encodinghuman or mouse PD-L3 OR VISTAPD-L3 OR VISTA OR VISTA disclosed herein,antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of PD-L3 ORVISTA mRNA, but more preferably is an oligonucleotide which is antisenseto only a portion of the coding or noncoding region of PD-L3 OR VISTAmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of PD-L3 OR VISTA ORVISTA mRNA. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid molecule of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid molecule (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridin-e,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiour-acil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a PD-L3 ORVISTAPD-L3 OR VISTA OR VISTA polypeptide to thereby inhibit expressionof the polypeptide, e.g., by inhibiting transcription and/ortranslation. The hybridization can be by conventional nucleotidecomplementarity to form a stable duplex, or, for example, in the case ofan antisense nucleic acid molecule which binds to DNA duplexes, throughspecific interactions in the major groove of the double helix. Anexample of a route of administration of antisense nucleic acid moleculesof the invention include direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an .alpha.-anomeric nucleic acid molecule. An.alpha.-anomeric nucleic acid molecule forms specific double-strandedhybrids with complementary RNA in which, contrary to the usual.beta.-units, the strands run parallel to each other (Gaultier et al.(1987) Nucleic Acids Res. 15:6625-6641). The antisense nucleic acidmolecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.(1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue(Inoue et al. (1987) FEBS Lett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach(1988) Nature 334:585-591)) can be used to catalytically cleave PD-L3 ORVISTA mRNA transcripts to thereby inhibit translation of PD-L3 OR VISTAOR VISTA mRNA. A ribozyme having specificity for a PD-L3 ORVISTA-encoding nucleic acid can be designed based upon the nucleotidesequence of a PD-L3 OR VISTA cDNA disclosed herein (i. e., SEQ ID NO: 1or 3). For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in a PD-L3 ORVISTAPD-L3 OR VISTA OR VISTA-encoding mRNA. See, e.g., Cech et al., U.S.Pat. No. 4,987,071 and Cech et al., U.S. Pat. No. 5,116,742.Alternatively, PD-L3 OR VISTA mRNA can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, PD-L3 or VISTA gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe PD-L3 or VISTA (e.g., the PD-L3 or VISTA promoter and/or enhancers;to form triple helical structures that prevent transcription of thePD-L3 gene in target cells. See generally, Helene, C (1991) AnticancerDrug Des. 6(6):569-84; Helene, C et al. (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J. (1992) Bioessays 14(12):807-15.

In yet another embodiment, the PD-L3 or VISTA nucleic acid molecules ofthe present invention can be modified at the base moiety, sugar moietyor phosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup, B. and Nielsen, P. E. (1996) Bioorg.Med. Chem. 4(1):5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g, DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup and Nielsen (1996) supra andPerry-O'Keefe et al. (1996) Proc Natl. Acad. Sci. USA 93:14670-675.

PNAs of PD-L3 OR VISTA nucleic acid molecules can be used in therapeuticand diagnostic applications. For example, PNASscan be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, for example, inducing transcription or translation arrest orinhibiting replication. PNAs of PD-L3 OR VISTA nucleic acid moleculescan also be used in the analysis of single base pair mutations in a gene(e.g., by PNA-directed PCR clamping); as ‘artificial restrictionenzymes’ when used in combination with other enzymes (e.g., S1 nucleases(Hyrup and Nielsen (1996) supra)); or as probes or primers for DNAsequencing or hybridization (Hyrup and Nielsen (1996) supra;Perry-O'Keefe et al. (1996) supra).

In another embodiment, PNAs of PD-L3 OR VISTA can be modified (e.g., toenhance their stability or cellular uptake), by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. For example, PNA-DNA chimeras of PD-L3 OR VISTA nucleic acidmolecules can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNAseH and DNA polymerases), to interact with the DNA portion while the PNAportion would provide high binding affinity and specificity. PNA-DNAchimeras can be linked using linkers of appropriate lengths selected interms of base stacking, number of bonds between the nucleobases, andorientation (Hyrup and Nielsen (1996) supra). The synthesis of PNA-DNAchimeras can be performed as described in Hyrup and Nielsen (1996) supraand Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17):3357-63. Forexample, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used as a bridge between the PNA and the 5′ endof DNA (Mag, M. et al. (1989) Nucleic Acids Res. 17:5973-88). PNAmonomers are then coupled in a stepwise manner to produce a chimericmolecule with a 5′ PNA segment and a 3′ DNA segment (Finn P. J. et al.(1996) supra). Alternatively, chimeric molecules can be synthesized witha 5′ DNA segment and a 3′ PNA segment (Peterser, K. H. et al. (1975)Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Biotechniques 6:958-976) or intercalating agents (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

Alternatively, the expression characteristics of an endogenous PD-L3 ORVISTA gene within a cell line or microorganism may be modified byinserting a heterologous DNA regulatory element into the genome of astable cell line or cloned microorganism such that the insertedregulatory element is operatively linked with the endogenous PD-L3 ORVISTA gene. For example, an endogenous PD-L3 OR VISTA gene which isnormally “transcriptionally silent”, i.e., a PD-L3 OR VISTA gene whichis normally not expressed, or is expressed only at very low levels in acell line or microorganism, may be activated by inserting a regulatoryelement which is capable of promoting the expression of a normallyexpressed gene product in that cell line or microorganism.Alternatively, a transcriptionally silent, endogenous PD-L3 OR VISTAgene may be activated by insertion of a promiscuous regulatory elementthat works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked with anendogenous PD-L3 OR VISTA gene, using techniques, such as targetedhomologous recombination, which are well known to those of skill in theart, and described, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCTpublication No. WO 91/06667, published May 16, 1991.

II. Isolated PD-L3 OR VISTA Polypeptides and Anti-PD-L3 OR VISTAAntibodies

One aspect of the invention pertains to isolated PD-L3 OR VISTApolypeptides, and biologically active portions thereof, as well aspolypeptide fragments suitable for use as immunogens to raise anti-PD-L3OR VISTA antibodies. In one embodiment, native PD-L3 OR VISTApolypeptides can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. In another embodiment, PD-L3 OR VISTA polypeptides areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a PD-L3 OR VISTA protein or polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

An “isolated” or “purified” polypeptide or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thePD-L3 OR VISTA polypeptide is derived, or substantially free fromchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof PD-L3 OR VISTA polypeptide in which the polypeptide is separated fromcellular components of the cells from which it is isolated orrecombinantly produced. In one embodiment, the language “substantiallyfree of cellular material” includes preparations of PD-L3 OR VISTApolypeptide having less than about 30% (by dry weight) of non-PD-L3 ORVISTA protein (also referred to herein as a “contaminating protein”),more preferably less than about 20% of non-PD-L3 OR VISTA protein, stillmore preferably less than about 10% of non-PD-L3 OR VISTA protein, andmost preferably less than about 5% non-PD-L3 OR VISTA protein. When thePD-L3 OR VISTA polypeptide or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of PD-L3 OR VISTA polypeptide in whichthe polypeptide is separated from chemical precursors or other chemicalswhich are involved in the synthesis of the polypeptide. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of PD-L3 OR VISTA polypeptidehaving less than about 30% (by dry weight) of chemical precursors ornon-PD-L3 OR VISTA chemicals, more preferably less than about 20%chemical precursors or non-PD-L3 OR VISTA chemicals, still morepreferably less than about 10% chemical precursors or non-PD-L3 OR VISTAchemicals, and most preferably less than about 5% chemical precursors ornon-PD-L3 OR VISTA chemicals.

As used herein, a “biologically active portion” of a PD-L3 OR VISTApolypeptide includes a fragment of a PD-L3 OR VISTA polypeptide whichparticipates in an interaction between a PD-L3 OR VISTA molecule and anon-PD-L3 OR VISTA molecule, e.g., a natural ligand of PD-L3 OR VISTA.Biologically active portions of a PD-L3 OR VISTA polypeptide includepeptides comprising amino acid sequences sufficiently identical to orderived from the amino acid sequence of the PD-L3 OR VISTA polypeptide,e.g., the amino acid sequence shown in SEQ ID NO: 2, 4 or 5, whichinclude fewer amino acids than the full length PD-L3 OR VISTApolypeptides, and exhibit at least one activity of a PD-L3 OR VISTApolypeptide. Typically, biologically active portions comprise a domainor motif with at least one activity of the PD-L3 OR VISTA polypeptide,e.g., modulating (suppressing)CD4 T cell proliferative responses toanti-CD3, suppression of the proliferative response of cognate CD4 Tcells in an antigen specific manner, effects on the expression ofspecific cytokines, et al. A biologically active portion of a PD-L3 ORVISTA polypeptide can be a polypeptide which is, for example, 25, 50,75, 100, 125, 150, 175, 200, 225 or more amino acids in length.Biologically active portions of a PD-L3 OR VISTA polypeptide can be usedas targets for developing agents which modulate a PD-L3 ORVISTA-mediated activity, e.g., immune cell activation.

In one embodiment, a biologically active portion of a PD-L3 OR VISTApolypeptide comprises at least a portion of an extracellular domain. Itis to be understood that a preferred biologically active portion of aPD-L3 OR VISTA polypeptide of the present invention may contain at leasta portion of an extracellular domain (e.g., comprising an IgV), and oneor more of the following domains: a signal peptide domain, atransmembrane domain, and a cytoplasmic domain. Moreover, otherbiologically active portions, in which other regions of the polypeptideare deleted, can be prepared by recombinant techniques and evaluated forone or more of the functional activities of a native PD-L3 OR VISTApolypeptide.

In a preferred embodiment, the PD-L3 OR VISTA polypeptide has an aminoacid sequence shown in SEQ ID NO: 2, 4 or 5. In other embodiments, thePD-L3 OR VISTA polypeptide is substantially identical to SEQ ID NO: 2, 4or 5, and retains the functional activity of the polypeptide of SEQ IDNO: 2, 4 or 5, yet differs in amino acid sequence due to natural allelicvariation or mutagenesis, as described above.

The nucleic acid and polypeptide sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to PD-L3 OR VISTA nucleic acid molecules of the invention.BLAST protein searches can be performed with the XBLAST program,score=100, wordlength=3 to obtain amino acid sequences homologous toPD-L3 OR VISTA polypeptide molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seethe internet website for the National Center for BiotechnologyInformation.

The invention also provides PD-L3 OR VISTA chimeric or fusion proteins.As used herein, a PD-L3 OR VISTA “chimeric protein” or “fusion protein”comprises a PD-L3 OR VISTA polypeptide operatively linked to a non-PD-L3OR VISTA polypeptide. A “PD-L3 OR VISTA polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a PD-L3 ORVISTA molecule, whereas a “non-PD-L3 OR VISTA polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a polypeptidewhich is not substantially homologous to the PD-L3 OR VISTA polypeptide,e.g., a polypeptide which is different from the PD-L3 OR VISTApolypeptide and which is derived from the same or a different organism.Within a PD-L3 OR VISTA fusion protein, the PD-L3 OR VISTA polypeptidecan correspond to all or a portion of a PD-L3 OR VISTA polypeptide. In apreferred embodiment, a PD-L3 OR VISTA fusion protein comprises at leastone biologically active portion of a PD-L3 OR VISTA polypeptide. Inanother preferred embodiment, a PD-L3 OR VISTA fusion protein comprisesat least two domains of a PD-L3 OR VISTA polypeptide. Within the fusionprotein, the term “operatively linked” is intended to indicate that thePD-L3 OR VISTA polypeptide and the non-PD-L3 OR VISTA polypeptide arefused in-frame to each other. The non-PD-L3 OR VISTA polypeptide can befused to the N-terminus or C-terminus of the PD-L3 OR VISTA polypeptideand corresponds to a moiety that alters the solubility, bindingaffinity, stability, or valency of the PD-L3 OR VISTA polypeptide.

For example, in one embodiment, the fusion protein is a GST-PD-L3 ORVISTA fusion protein in which the PD-L3 OR VISTA sequences are fused tothe C-terminus of the GST sequences. Such fusion proteins can facilitatethe purification of recombinant PD-L3 OR VISTA. In another embodiment,the fusion protein is a PD-L3 OR VISTA polypeptide containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of PD-L3 ORVISTA can be increased through use of a heterologous signal sequence. Ina preferred embodiment, the fusion protein is an Ig-PD-L3 OR VISTAfusion protein in which the PD-L3 OR VISTA sequences are fused to aportion of an Ig molecule. The Ig portion of the fusion protein caninclude and immunoglobulin constant region, e.g., a human Cgamma1 domainor a C gamma4 domain (e.g., the hinge, CH2, and CH3 regions of human IgCgamma1 or human IgC gamma4 (see, e.g., Capon et al., U.S. Pat. Nos.5,116,964; 5,580,756; 5,844,095, and the like, incorporated herein byreference). A resulting fusion protein may have altered PD-L3 OR VISTAsolubility, binding affinity, stability and/or valency (i.e., the numberof binding sites per molecule) and may increase the efficiency ofprotein purification.

Particularly preferred PD-L3 OR VISTA Ig fusion proteins include anextracellular domain portion of PD-L3 OR VISTA coupled to animmunoglobulin constant region (e.g, the Fc region). The immunoglobulinconstant region may contain genetic modifications which reduce oreliminate effector activity inherent in the immunoglobulin structure.For example, DNA encoding an extracellular portion of a PD-L3 OR VISTApolypeptide can be joined to DNA encoding the hinge, CH2, and CH3regions of human IgG gamma1 and/or IgG gamma4 modified by site-directedmutagenesis, e.g., as taught in WO 97/28267. The PD-L3 OR VISTA fusionproteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject in vivo. The PD-L3 OR VISTAfusion proteins can be used to affect the bioavailability of a PD-L3 ORVISTA binding partner. Use of PD-L3 OR VISTA fusion proteins may beuseful therapeutically for the treatment of conditions or disorders thatwould benefit from modulation of the immune response. Moreover, thePD-L3 OR VISTA-fusion proteins of the invention can be used asimmunogens to produce anti-PD-L3 OR VISTA antibodies in a subject, topurify PD-L3 OR VISTA-binding proteins, and in screening assays toidentify molecules which inhibit the interaction of PD-L3 OR VISTA withits natural binding partner, 001941 Preferably, a PD-L3 OR VISTAchimeric or fusion protein of the invention is produced by standardrecombinant DNA techniques.

The present invention also pertains to variants of the PD-L3 OR VISTApolypeptides which function as either PD-L3 OR VISTA agonists (mimetics)or as PD-L3 OR VISTA antagonists. Variants of the PD-L3 OR VISTApolypeptides can be generated by mutagenesis, e.g., discrete pointmutation or truncation of a PD-L3 OR VISTA polypeptide. An agonist ofthe PD-L3 OR VISTA polypeptides can retain substantially the same, or asubset, of the biological activities of the naturally occurring form ofa PD-L3 OR VISTA polypeptide. An antagonist of a PD-L3 OR VISTApolypeptide can inhibit one or more of the activities of the naturallyoccurring form of the PD-L3 OR VISTA polypeptide by, for example,competitively modulating a PD-L3 OR VISTA-mediated activity of a PD-L3OR VISTA polypeptide. Thus, specific biological effects can be elicitedby treatment with a variant of limited function. In one embodiment,treatment of a subject with a variant having a subset of the biologicalactivities of the naturally occurring form of the polypeptide has fewerside effects in a subject relative to treatment with the naturallyoccurring form of the PD-L3 OR VISTA polypeptide.

In one embodiment, variants of a PD-L3 OR VISTA polypeptide whichfunction as either PD-L3 OR VISTA agonists (mimetics) or as PD-L3 ORVISTA antagonists can be identified by screening combinatorial librariesof mutants, e.g., truncation mutants, of a PD-L3 OR VISTA polypeptidefor PD-L3 OR VISTA polypeptide agonist or antagonist activity. In oneembodiment, a variegated library of PD-L3 OR VISTA variants is generatedby combinatorial mutagenesis at the nucleic acid level and is encoded bya variegated gene library. A variegated library of PD-L3 OR VISTAvariants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential PD-L3 OR VISTA sequences is expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of PD-L3 OR VISTAsequences therein. There are a variety of methods which can be used toproduce libraries of potential PD-L3 OR VISTA variants from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential PD-L3 ORVISTA sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477).

In addition, libraries of fragments of a PD-L3 OR VISTA polypeptidecoding sequence can be used to generate a variegated population of PD-L3OR VISTA fragments for screening and subsequent selection of variants ofa PD-L3 OR VISTA polypeptide. In one embodiment, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of a PD-L3 OR VISTA coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PD-L3 OR VISTApolypeptide.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of PD-L3 OR VISTApolypeptides. The most widely used techniques, which are amenable tohigh through-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify PD-L3 OR VISTA variants (Arkin and Youvan(1992) Proc Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993)Protein Eng. 6(3):327-331).

In addition to PD-L3 OR VISTA polypeptides consisting only ofnaturally-occurring amino acids, PD-L3 OR VISTA peptidomimetics are alsoprovided. Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. These types of non-peptide compounds are termed“peptide mimetics” or “peptidomimetics” (Fauchere, J. (1986) Adv. DrugRes. 15:29; Veber and Freidinger (1985) TINS p. 392; and Evans et al.(1987) J. Med. Chem 30:1229, which are incorporated herein by reference)and are usually developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar totherapeutically useful peptides can be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i e., a polypeptide thathas a biological or pharmacological activity), such as human or mousePD-L3 OR VISTA, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: —CH2NH—,—CH2S—, —CH2-CH2-, —CH.dbd.CH— (cis and trans), —COCH2-, —CH(OH)CH2-,and —CH2SO—, by methods known in the art and further described in thefollowing references: Spatola, A. F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins Weinstein, B., ed., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, “Peptide Backbone Modifications”; Morley, J. S. (1980) Trends.Pharm. Sci. pp. 463-468; Hudson, D. et al. (1979) Int. J. Pept. Prot.Res. 14:177-185 (—CH2NH—, CH2CH2-); Spatola, A. F. et al. (1986) Life.Sci. 38:1243-1249 (—CH2-S); Hann, M. M. (1982) J. Chem. SoC Perkin.Trans. I 307-314 (—CH—CH—, cis and trans); Almquist, R. G. et al. (1980)J. Med. Chem. 23:1392-1398 (—COCH2-); Jennings-White, C et al. (1982)Tetrahedron Lett. 23:2533 (—COCH2-); Szelke, M. et al., European PatentApplication No. EP 45665 (1982) CA: 97:39405 (—CH(OH)CH2-); Holladay, M.W. et al. (1983) Tetrahedron. Lett. 24:4401-4404 (—C(OH)CH2-); andHruby, V. J. (1982) Life Sci. 31:189-199 (—CH2-S—); each of which isincorporated herein by reference. A particularly preferred non-peptidelinkage is —CH2NH—. Such peptide mimetics may have significantadvantages over polypeptide embodiments, including, for example: moreeconomical production, greater chemical stability, enhancedpharmacological properties (half-life, absorption, potency, efficacy,etc.), altered specificity (e.g., a broad-spectrum of biologicalactivities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) to which the peptidomimeticbinds to produce the therapeutic effect. Derivitization (e.g., labeling)of peptidomimetics should not substantially interfere with the desiredbiological or pharmacological activity of the peptidomimetiC

Systematic substitution of one or more amino acids of a PD-L3 OR VISTAamino acid sequence with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) can be used to generate more stable peptides. Inaddition, constrained peptides comprising a PD-L3 OR VISTA amino acidsequence or a substantially identical sequence variation can begenerated by methods known in the art (Rizo and Gierasch (1992) Annu.Rev. Biochem. 61:387, incorporated herein by reference); for example, byadding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide. The amino acid sequences ofthe PD-L3 OR VISTA polypeptides identified herein will enable those ofskill in the art to produce polypeptides corresponding to PD-L3 OR VISTApeptide sequences and sequence variants thereof. Such polypeptides canbe produced in prokaryotic or eukaryotic host cells by expression ofpolynucleotides encoding a PD-L3 OR VISTA peptide sequence, frequentlyas part of a larger polypeptide. Alternatively, such peptides can besynthesized by chemical methods. Methods for expression of heterologouspolypeptides in recombinant hosts, chemical synthesis of polypeptides,and in vitro translation are well known in the art. Certainamino-terminal and/or carboxy-terminal modifications and/or peptideextensions to the core sequence can provide advantageous physical,chemical, biochemical, and pharmacological properties, such as: enhancedstability, increased potency and/or efficacy, resistance to serumproteases, desirable pharmacokinetic properties, and others. Peptidescan be used therapeutically to treat disease, e.g., by alteringcostimulation in a patient.

An isolated PD-L3 OR VISTA polypeptide, or a portion or fragmentthereof, can be used as an immunogen to generate antibodies that bindPD-L3 OR VISTA using standard techniques for polyclonal and monoclonalantibody preparation. A full-length PD-L3 OR VISTA polypeptide can beused or, alternatively, the invention provides antigenic peptidefragments of PD-L3 OR VISTA for use as immunogens. In one embodiment, anantigenic peptide of PD-L3 OR VISTA comprises at least 8 amino acidresidues of the amino acid sequence shown in SEQ ID NO: 2, 4 or 5 andencompasses an epitope of PD-L3 OR VISTA such that an antibody raisedagainst the peptide forms a specific immune complex with the PD-L3 ORVISTA polypeptide. Preferably, the antigenic peptide comprises at least10 amino acid residues, more preferably at least 15 amino acid residues,even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of PD-L3 OR VISTA thatare located in the extracellular domain of the polypeptide, e.g.,hydrophilic regions, as well as regions with high antigenicity.

A PD-L3 OR VISTA immunogen typically is used to prepare antibodies byimmunizing a suitable subject (e.g., rabbit, goat, mouse, or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed PD-L3 OR VISTA polypeptideor a chemically synthesized PD-L3 OR VISTA polypeptide. The preparationcan further include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory agent. Immunization of a suitablesubject with an immunogenic PD-L3 OR VISTA preparation induces apolyclonal anti-PD-L3 OR VISTA antibody response.

Accordingly, another aspect of the invention pertains to anti-PD-L3 ORVISTA antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as a PD-L3 OR VISTA. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)2 fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind PD-L3OR VISTA molecules. The term “monoclonal antibody” or “monoclonalantibody composition”, as used herein, refers to a population ofantibody molecules that contain only one species of an antigen bindingsite capable of immunoreacting with a particular epitope of PD-L3 ORVISTA. A monoclonal antibody composition thus typically displays asingle binding affinity for a particular PD-L3 OR VISTA polypeptide withwhich it immunoreacts.

Polyclonal anti-PD-L3 OR VISTA antibodies can be prepared as describedabove by immunizing a suitable subject with a PD-L3 OR VISTA immunogen,e.g., a PD-L3 OR VISTA-Ig fusion protein. The anti-PD-L3 OR VISTAantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized PD-L3 OR VISTA. If desired, the antibodymolecules directed against PD-L3 OR VISTA can be isolated from themammal (e.g., from the blood) and further purified by well knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g, when the anti-PD-L3 ORVISTA antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein (1975) Nature 256:495-497 (see alsoBrown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol.Chem. 255:4980-83; Yeh et al. (1976) Proc Natl. Acad. Sci. USA76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the morerecent human B cell hybridoma technique (Kozbor et al. (1983) Immunol.Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or triomatechniques. The technology for producing monoclonal antibody hybridomasis well known (see generally Kenneth, R. H. in Monoclonal Antibodies: ANew Dimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); Lemer, E. A. (1981) Yale J. Biol. Med. 54:387-402; Gefter,M. L. et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortalcell line (typically a myeloma) is fused to lymphocytes (typicallysplenocytes) from a mammal immunized with a PD-L3 OR VISTA immunogen asdescribed above, and the culture supernatants of the resulting hybridomacells are screened to identify a hybridoma producing a monoclonalantibody that binds PD-L3 OR VISTA. Any of the many well known protocolsused for fusing lymphocytes and immortalized cell lines can be appliedfor the purpose of generating an anti-PD-L3 OR VISTA monoclonal antibody(see, e.g., Galfre, G. et al. (1977) Nature 266:55052; Gefter et al.(1977) supra; Lerner (1981) supra; and Kenneth (1980) supra). Moreover,the ordinarily skilled worker will appreciate that there are manyvariations of such methods which also would be useful. Typically, theimmortal cell line (e.g., a myeloma cell line) is derived from the samemammalian species as the lymphocytes. For example, murine hybridomas canbe made by fusing lymphocytes from a mouse immunized with an immunogenicpreparation of the present invention with an immortalized mouse cellline. Preferred immortal cell lines are mouse myeloma cell lines thatare sensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind PD-L3 OR VISTA, e.g., using a standard ELISAassay.

Specific methods for producing antibodies that bind PD-L3 OR VISTA canbe effected using methods known in the art and as described in theexamples. Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-PD-L3 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with PD-L3 OR VISTA to therebyisolate immunoglobulin library members that bind PD-L3 OR VISTA. Kitsfor generating and screening phage display libraries are commerciallyavailable

As noted these antibodies re screened to identify those that bind tospecific epitopes of PD-L3 OR VISTA, e.g. in the IgV domain or otherspecific domains and/or to select antibodies possessing high affinityand avidity to PD-L3 OR VISTA protein. In addition these antibodies arescreened to identify those of which modulate specific functions andeffects of PD-L3 OR VISTA on immunity and immune cells in vitro and invivo. For example assays can be conducted to ascertain the modulatoryeffect, if any, of a particular anti-PD-L3 OR VISTA antibody on immunefunctions negatively regulated by PD-L3 OR VISTA including cytokineproduction by CD4+ or CD8+ T cells, CD28 costimulation, CD4+ T cellproliferation, and the proliferation of naïve and memory CD4+ T cells,et al. In a preferred embodiment assays are conducted to identifypotential therapeutic anti-PD-L3 OR VISTA antibodies which in vitro,when the presence of PD-L3 OR VISTA-Ig enhance the suppression by PD-L3OR VISTA-Ig as these anti-PD-L3 OR VISTA antibodies behave oppositely invivo, i.e., they are immunosuppressive. The invention encompassesanti-VISTA antibodies and use thereof that specifically bind to the 136amino acid extracellular domain, e.g., to amino acids 1-50, 50-100,100-136, antibodies that specifically bind the IgV, antibodies thatspecifically bind the stalk region, antibodies that specifically bindthe transmembrane region and antibodies that specifically bind thecytoplasmic region of VISTA. These specific regions are identified inthe application.

Additionally, recombinant anti-PD-L3 OR VISTA antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al., International Application No. PCT/US86/02269; Akira etal., European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al., European Patent Application173,494; Neuberger et al., PCT International Publication No. WO86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc Natl. Acad. Sci. USA 84:3439-3443;Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) ProcNatl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cancer Res.47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988)J. Natl. Cancer Inst. 80:1553-1559; Morrison, S. L. (1985) Science229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter, U.S. Pat.No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyen et al.(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

An anti-PD-L3 OR VISTA antibody (e.g., monoclonal antibody) can be usedto isolate PD-L3 OR VISTA by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-PD-L3 OR VISTA antibodycan facilitate the purification of natural PD-L3 OR VISTA from cells andof recombinantly produced PD-L3 OR VISTA expressed in host cells.Moreover, an anti-PD-L3 OR VISTA antibody can be used to detect PD-L3 ORVISTA polypeptide (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of the PD-L3OR VISTA polypeptide. Anti-PD-L3 OR VISTA antibodies can be useddiagnostically to monitor polypeptide levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,.beta.-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include 125I, 131I, 35S or3H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid molecule encoding a PD-L3OR VISTA polypeptide (or a portion thereof). As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) Methods Enzymol. 185:3-7.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cells and those whichdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., PD-L3 OR VISTApolypeptides, mutant forms of PD-L3 OR VISTA polypeptides, fusionproteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of PD-L3 OR VISTA polypeptides in prokaryotic or eukaryoticcells. For example, PD-L3 OR VISTA polypeptides can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors), yeast cells, or mammalian cells. Suitable hostcells are discussed further in Goeddel (1990) supra. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase. Purified fusion proteins can be utilized in PD-L3 OR VISTAactivity assays (e.g., direct assays or competitive assays described indetail below), or to generate antibodies specific for PD-L3 OR VISTApolypeptides, for example. In another embodiment, the PD-L3 OR VISTAexpression vector is a yeast expression vector. Examples of vectors forexpression in yeast S. cerevisiae include pYepSecl (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp,San Diego, Calif.). Alternatively, PD-L3 OR VISTA polypeptides can beexpressed in insect cells using baculovirus expression vectors.Baculovirus vectors available for expression of polypeptides in culturedinsect cells (e.g., Sf9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39). In yet another embodiment, a nucleicacid of the invention is expressed in mammalian cells using a mammalianexpression vector. Examples of mammalian expression vectors includepCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)EMBO J. 6:187-195). When used in mammalian cells, the expressionvector's control functions are often provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitableexpression systems for both prokaryotic and eukaryotic cells seechapters 16 and 17 of Sambrook, J. et al., Molecular Cloning: ALaboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example by the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the .alpha.-fetoprotein promoter (Campesand Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PD-L3 OR VISTA mRNA. Regulatory sequencesoperatively linked to a nucleic acid molecule cloned in the antisenseorientation can be chosen which direct the continuous expression of theantisense RNA molecule in a variety of cell types, for instance viralpromoters and/or enhancers, or regulatory sequences can be chosen whichdirect constitutive, tissue specific, or cell type specific expressionof antisense RNA. The antisense expression vector can be in the form ofa recombinant plasmid, phagemid, or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes, see Weintraub, H.et al., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which aPD-L3 OR VISTA nucleic acid molecule of the invention is introduced,e.g., a PD-L3 OR VISTA nucleic acid molecule within a recombinantexpression vector or a PD-L3 OR VISTA nucleic acid molecule containingsequences which allow it to homologously recombine into a specific siteof the host cell's genome. The terms “host cell” and “recombinant hostcell” are used interchangeably herein. It is understood that such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A host cell can be any prokaryotic or eukaryotic cell. VectorDNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals. In order to identify and select theseintegrants, a gene that encodes a selectable marker (e.g., resistance toantibiotics) is generally introduced into the host cells along with thegene of interest. Preferred selectable markers include those whichconfer resistance to drugs, such as G418, hygromycin and methotrexate. Ahost cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a PD-L3 OR VISTApolypeptide. Accordingly, the invention further provides methods forproducing a PD-L3 OR VISTA polypeptide using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of the invention (into which a recombinant expression vectorencoding a PD-L3 OR VISTA polypeptide has been introduced) in a suitablemedium such that a PD-L3 OR VISTA polypeptide is produced. In anotherembodiment, the method further comprises isolating a PD-L3 OR VISTApolypeptide from the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichPD-L3 OR VISTA-coding sequences have been introduced. Such host cellscan then be used to create non-human transgenic animals in whichexogenous PD-L3 OR VISTA sequences have been introduced into theirgenome or homologous recombinant animals in which endogenous PD-L3 ORVISTA sequences have been altered. Such animals are useful for studyingthe function and/or activity of a PD-L3 OR VISTA and for identifyingand/or evaluating modulators of PD-L3 OR VISTA activity. As used herein,a “transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous PD-L3 OR VISTA gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal. Atransgenic animal of the invention can be created by introducing a PD-L3OR VISTA-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The PD-L3 ORVISTA cDNA sequence of SEQ ID NO: 1 or 4 can be introduced as atransgene into the genome of a non-human animal. Alternatively, anonhuman homologue of a human PD-L3 OR VISTA gene, such as a monkey orrat PD-L3 OR VISTA gene, can be used as a transgene. Alternatively, aPD-L3 OR VISTA gene homologue, such as another PD-L3 OR VISTA familymember, can be isolated based on hybridization to the PD-L3 OR VISTAcDNA sequences of SEQ ID NO: 1, or 3 (described further in subsection Iabove) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to a PD-L3 OR VISTA transgene to directexpression of a PD-L3 OR VISTA polypeptide to particular cells. Methodsfor generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a PD-L3 OR VISTA transgene in its genome and/or expressionof PD-L3 OR VISTA mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encoding aPD-L3 OR VISTA polypeptide can further be bred to other transgenicanimals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a PD-L3 OR VISTA gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the PD-L3 OR VISTA gene. The PD-L3 OR VISTAgene can be a human or murine gene (e.g., the cDNA of SEQ ID NO: 1 or 3)

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc Natl. Acad. Sci.USA 89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. (1991) Science251:1351-1355. If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected polypeptide are required. Suchanimals can be provided through the construction of “double” transgenicanimals, e.g., by mating two transgenic animals, one containing atransgene encoding a selected polypeptide and the other containing atransgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter GO phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to themorula or blastocyst stage and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

IV. Pharmaceutical Compositions

The PD-L3 OR VISTA molecules, e.g, the PD-L3 OR VISTA nucleic acidmolecules, fragments of PD-L3 OR VISTA polypeptides, and anti-PD-L3 ORVISTA antibodies (also referred to herein as “active compounds” or“modulating agents”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, polypeptide,or antibody and a carrier, e.g., a pharmaceutically acceptable carrier.As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

As noted such compositions may additionally comprise a desired antigen,e.g., a tumor antigen or another immune modulatory compounds such asToll like receptor agonists, type 1 interferon such as alpha and betainterferons and CD40 agonists such as agonistic CD40 antibodies andantibody fragments, preferably anti-human CD40 agonistic antibodies andantibody fragments or other immune enhancers or suppressors such asPD-L1, PD-L2, CTLA4 fusion proteins and antibodies specific thereto.

In some preferred embodiments, the composition or PD-L3 OR VISTA basedtherapy may further include an antigen or other immune agonist. Whenpresent in the composition or therapy, the antigen may be administeredin an amount that, in combination with the other components of thecombination, is effective to generate an immune response against theantigen. For example, the antigen can be administered in an amount fromabout 100 .mu.g/kg to about 100 mg/kg. In some embodiments, the antigenmay be administered in an amount from about 10 .mu.g/kg to about 10mg/kg. In some embodiments, the antigen may be administered in an amountfrom about 1 mg/kg to about 5 mg/kg. The particular amount of antigenthat constitutes an amount effective to generate an immune response,however, depends to some extent upon certain factors such as, forexample, the particular antigen being administered; the particularagonist being administered and the amount thereof; the particularagonist being administered and the amount thereof; the state of theimmune system; the method and order of administration of the agonist andthe antigen; the species to which the formulation is being administered;and the desired therapeutic result. Accordingly, it is not practical toset forth generally the amount that constitutes an effective amount ofthe antigen. Those of ordinary skill in the art, however, can readilydetermine the appropriate amount with due consideration of such factors.

The antigen can be any material capable of raising a Th1 immuneresponse, which may include one or more of, for example, a CD8+ T cellresponse, an NK T cell response, a .gamma./.delta. T cell response, or aTh1 antibody response. Suitable antigens include but are not limited topeptides; polypeptides; lipids; glycolipids; polysaccharides;carbohydrates; polynucleotides; prions; live or inactivated bacteria,viruses or fungi; and bacterial, viral, fungal, protozoal,tumor-derived, or organism-derived antigens, toxins or toxoids.

Furthermore, certain currently experimental antigens, especiallymaterials such as recombinant proteins, glycoproteins, and peptides thatdo not raise a strong immune response, can be used in connection withadjuvant combinations of the invention. Exemplary experimental subunitantigens include those related to viral disease such as adenovirus,AIDS, chicken pox, cytomegalovirus, dengue, feline leukemia, fowlplague, hepatitis A, hepatitis B, HSV-1, HSV-2, hog cholera, influenzaA, influenza B, Japanese encephalitis, measles, parainfluenza, rabies,respiratory syncytial virus, rotavirus, wart, and yellow fever.

In one embodiment, the antigen may be a cancer antigen or a tumorantigen. The terms cancer antigen and tumor antigen are usedinterchangeably and refer to an antigen that is differentially expressedby cancer cells. Therefore, cancer antigens can be exploited todifferentially target an immune response against cancer cells. Cancerantigens may thus potentially stimulate tumor-specific immune responses.Certain cancer antigens are encoded, though not necessarily expressed,by normal cells. Some of these antigens may be characterized as normallysilent (i.e., not expressed) in normal cells, those that are expressedonly at certain stages of differentiation, and those that are temporallyexpressed (e.g., embryonic and fetal antigens). Other cancer antigenscan be encoded by mutant cellular genes such as, for example, oncogenes(e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), orfusion proteins resulting from internal deletions or chromosomaltranslocations. Still other cancer antigens can be encoded by viralgenes such as those carried by RNA and DNA tumor viruses.

Examples of tumor antigens include MAGE, MART-1/Melan-A, gp100,Dipeptidyl peptidase 1V (DPPUV), adenosine deaminase-binding protein(ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its antigenicepitopes CAP-1 and CAP-2, etv6, am11, Prostate Specific Antigen (PSA)and its antigenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-.zeta. chain, MAGE-familyof tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family, HER2/neu, p21ras, RCAS1, .alpha.-fetoprotein,.epsilon.-cadherin, .alpha.-catenin, .beta.-catenin, .gamma.-catenin,p120ctn, gp10.sup.Pmell 17, PRAME, NY-ESO-1, cdc27, adenomatouspolyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15,gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, Smad family of tumor antigens, Imp-1, PIA, EBV-encodednuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.

Cancers or tumors and specific tumor antigens associated with suchtumors (but not exclusively), include acute lymphoblastic leukemia(etv6, aml1, cyclophilin b), B cell lymphoma (Ig-idiotype), glioma(E-cadherin, .alpha.-catenin, .beta.-catenin, .gamma.-catenin, p120ctn),bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUCfamily, HER2/neu, c-erbB-2), cervical carcinoma (p53, p21ras), coloncarcinoma (p21ras, HER2/neu, c-erbB-2, MUC family), colorectal cancer(Colorectal associated antigen (CRC)-CO17-1A/GA733, APC),choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b), gastriccancer (HER2/neu, c-erbB-2, ga733 glycoprotein), hepatocellular cancer(.alpha.-fetoprotein), Hodgkins lymphoma (Imp-1, EBNA-1), lung cancer(CEA, MAGE-3, NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b),melanoma (p5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides,Melan-A/MART-1, cdc27, MAGE-3, p21ras, gp100.sup.Pmel117), myeloma (MUCfamily, p21ras), non-small cell lung carcinoma (HER2/neu, c-erbB-2),nasopharyngeal cancer (Imp-1, EBNA-1), ovarian cancer (MUC family,HER2/neu, c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA)and its antigenic epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu,c-erbB-2, ga733 glycoprotein), renal cancer (HER2/neu, c-erbB-2),squamous cell cancers of the cervix and esophagus (viral products suchas human papilloma virus proteins), testicular cancer (NY-ESO-1), and Tcell leukemia (HTLV-1 epitopes).

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., modulating agents such as a PD-L3 OR VISTA nucleic acidmolecule, a fragment of a PD-L3 OR VISTA polypeptide, an anti-PD-L3 ORVISTA antibody, or a combination of an anti-PD-L3 OR VISTA antibody andan anti-PD-L1 antibody) in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery. In one embodiment,the active compounds are prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, InC Liposomal suspensions (including liposomes targetedto infected cells with monoclonal antibodies to viral antigens) can alsobe used as pharmaceutically acceptable carriers. These can be preparedaccording to methods known to those skilled in the art, for example, asdescribed in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals. The data obtained from the cell culture assays and animalstudies can be used in formulating a range of dosage for use in humans.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity of PD-L3 OR VISTA. An agent may, for example, be a smallmolecule. For example, such small molecules include, but are not limitedto, peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds. It is understood that appropriatedoses of small molecule agents depends upon a number of factors withinthe scope of knowledge of the ordinarily skilled physician,veterinarian, or researcher. The dose(s) of the small molecule willvary, for example, depending upon the identity, size, and condition ofthe subject or sample being treated, further depending upon the route bywhich the composition is to be administered, if applicable, and theeffect which the practitioner desires the small molecule to have uponthe nucleic acid or polypeptide of the invention.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram). It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such polypeptides may include, for example, a toxin such asabrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a proteinsuch as tumor necrosis factor, alpha-interferon, beta-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors. Techniques for conjugating such therapeutic moiety toantibodies are well known.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see, e.g., Chen et al. (1994) Proc Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system. The pharmaceutical compositions can beincluded in a container, pack, or dispenser together with instructionsfor administration.

V. Uses and Methods of the Invention

The PD-L3 OR VISTA molecules, e.g., the PD-L3 OR VISTA nucleic acidmolecules, polypeptides, polypeptide homologues, and antibodies andantibody fragments described herein can be used in one or more of thefollowing methods: a) screening assays; b) predictive medicine (e.g.,diagnostic assays, prognostic assays, and monitoring clinical trials);and c) methods of treatment (e.g., therapeutic and prophylactic, e.g.,by up- or down-modulating the immune response). As described herein, aPD-L3 OR VISTA polypeptide of the invention has one or more of thefollowing activities: 1) binds to and/or modulates the activity of itsnatural binding partner(s), 2) modulates intra- or intercellularsignaling, 3) modulates activation of T lymphocytes, 4) modulates theimmune response of an organism, e.g., a mammalian organism, such as amouse or human. The isolated nucleic acid molecules of the invention canbe used, for example, to express PD-L3 OR VISTA polypeptide (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect PD-L3 OR VISTA mRNA (e.g., in a biologicalsample) or a genetic alteration in a PD-L3 OR VISTA gene, and tomodulate PD-L3 OR VISTA activity, as described further below. The PD-L3OR VISTA polypeptides can be used to treat conditions or disorderscharacterized by insufficient or excessive production of a PD-L3 ORVISTA polypeptide or production of PD-L3 OR VISTA inhibitors. Inaddition, the PD-L3 OR VISTA polypeptides can be used to screen fornaturally occurring PD-L3 OR VISTA binding partner(s), to screen fordrugs or compounds which modulate PD-L3 OR VISTA activity, as well as totreat conditions or disorders characterized by insufficient or excessiveproduction of PD-L3 OR VISTA polypeptide or production of PD-L3 OR VISTApolypeptide forms which have decreased, aberrant or unwanted activitycompared to PD-L3 OR VISTA wild-type polypeptide (e.g., immune systemdisorders such as severe combined immunodeficiency, multiple sclerosis,systemic lupus erythematosus, type I diabetes mellitus,lymphoproliferative syndrome, inflammatory bowel disease, allergies,asthma, graft-versus-host disease, and transplant rejection; immuneresponses to infectious pathogens such as bacteria and viruses; andimmune system cancers such as lymphomas and leukemias). Moreover, theanti-PD-L3 OR VISTA antibodies of the invention can be used to detectand isolate PD-L3 OR VISTA polypeptides, regulate the bioavailability ofPD-L3 OR VISTA polypeptides, and modulate PD-L3 OR VISTA activity, e.g.,by modulating the interaction between PD-L3 OR VISTA and its naturalbinding partner(s)

A. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to PD-L3 OR VISTA polypeptides, have a stimulatory orinhibitory effect on, for example, PD-L3 OR VISTA expression or PD-L3 ORVISTA activity, or have a stimulatory or inhibitory effect on theinteraction between PD-L3 OR VISTA and its natural binding partner(s).

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to the PD-L3 OR VISTA protein orpolypeptide or biologically active portion thereof, e.g., modulate theability of the PD-L3 OR VISTA polypeptide to interact with its naturalbinding partner(s). In another embodiment, the invention provides assaysfor screening candidate or test compounds which bind to or modulate theactivity of a PD-L3 OR VISTA protein or polypeptide or biologicallyactive portion thereof. In a preferred embodiment, the inventionprovides assays for screening candidate or test compounds which have astimulatory or inhibitory effect on immune functions negativelyregulated by PD-L3 OR VISTA such as are identified herein or based onits effect on the interaction of between PD-L3 OR VISTA and its naturalbinding partner(s). These PD-L3 OR VISTA related functions include byway of example inhibiting cytokine production (e.g., 11-2, gammainterferon by T cells, suppressing moderate CD28 costimulation,inhibiting CD4+ and CD8+ T cell proliferation, suppressing proliferationof naïve and memory CD4+ T cells, and suppressing TCR activation withoutinducing apoptosis. The test compounds of the present invention can beobtained using any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam, K. S. (1997) Anticancer Drug Des. 12:145).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a PD-L3 OR VISTA polypeptide or biologically active portionthereof is contacted with a test compound, and the ability of the testcompound to modulate PD-L3 OR VISTA activity is determined. Determiningthe ability of the test compound to modulate PD-L3 OR VISTA activity canbe accomplished by monitoring, for example, the ability of PD-L3 ORVISTA to bind to its natural binding partner(s), and modulate immunecell activity. The immune cell can be, e.g., a T cell, a B cell, or amyeloid cell. Determining the ability of the test compound to modulatePD-L3 OR VISTA binding to its counter-receptor (to be determined) can beaccomplished, for example, by coupling PD-L3 OR VISTA with aradioisotope or enzymatic label to monitor the ability of a testcompound to modulate PD-L3 OR VISTA binding to T cells which express thePD-L3 OR VISTA counter-receptor. Determining the ability of the testcompound to bind PD-L3 OR VISTA can be accomplished, for example, bycoupling the compound with a radioisotope or enzymatic label such thatbinding of the compound to PD-L3 OR VISTA can be determined by detectingthe labeled PD-L3 OR VISTA compound in a complex.

It is also within the scope of this invention to determine the abilityof a compound to interact with PD-L3 OR VISTA without the labeling ofany of the interactants. For example, a microphysiometer can be used todetect the interaction of a compound with PD-L3 OR VISTA without thelabeling of either the compound or the PD-L3 OR VISTA (McConnell, H. M.et al. (1992) Science 257:1906-1912). As used herein, a“microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and PD-L3 OR VISTA.

In another embodiment, an assay is a cell-based assay comprisingcontacting a T cell expressing a PD-L3 OR VISTA binding partner with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the PD-L3 OR VISTAbinding partner. Determining the ability of the test compound tomodulate the activity of a PD-L3 OR VISTA binding partner can beaccomplished, for example, by determining the ability of the PD-L3 ORVISTA polypeptide to bind to or interact with the PD-L3 OR VISTA bindingpartner.

Determining the ability of the PD-L3 OR VISTA polypeptide, or abiologically active fragment thereof, to bind to or interact with aPD-L3 OR VISTA binding partner, can be accomplished by one of themethods described above for determining direct binding. In a preferredembodiment, determining the ability of the PD-L3 OR VISTA polypeptide tobind to or interact with a PD-L3 OR VISTA binding partner can beaccomplished by determining the activity of the binding partner. Forexample, the activity of the binding partner can be determined bydetecting induction of a cellular second messenger (e.g., tyrosinekinase or phosphatase activity), detecting catalytic/enzymatic activityof an appropriate substrate, detecting the induction of a reporter gene(comprising a target-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a target-regulated cellular response. For example, determiningthe ability of the PD-L3 OR VISTA polypeptide to bind to or interactwith a natural PD-L3 OR VISTA binding partner, can be accomplished bymeasuring the ability of a compound to modulate immune cellcostimulation or inhibition in a proliferation assay, or by interferingwith the ability of a PD-L3 OR VISTA polypeptide to bind to antibodiesthat recognize a portion of the PD-L3 OR VISTA polypeptide. In oneembodiment, compounds that modulate T cell activation can be identifiedby determining the ability of a compound to modulate T cellproliferation or cytokine production. In a preferred embodiment,compounds that modulate T cell activation can be identified bydetermining the ability of a compound to modulate T cell proliferationor cytokine production at more than one antigen concentration.

In yet another embodiment, an assay of the present invention is acell-free assay in which a PD-L3 OR VISTA polypeptide or biologicallyactive portion thereof is contacted with a test compound and the abilityof the test compound to bind to the PD-L3 OR VISTA polypeptide orbiologically active portion thereof is determined. Preferredbiologically active portions of the PD-L3 OR VISTA polypeptides to beused in assays of the present invention include fragments whichparticipate in interactions with non-PD-L3 OR VISTA molecules, e.g., atleast a portion of an extracellular domain which binds to a PD-L3 ORVISTA binding partner. Binding of the test compound to the PD-L3 ORVISTA polypeptide can be determined either directly or indirectly asdescribed above.

In another embodiment, the assay is a cell-free assay in which a PD-L3OR VISTA polypeptide or biologically active portion thereof is contactedwith a test compound and the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the PD-L3 OR VISTApolypeptide or biologically active portion thereof is determined.Determining the ability of the test compound to modulate the activity ofa PD-L3 OR VISTA polypeptide can be accomplished, for example, bydetermining the ability of the PD-L3 OR VISTA polypeptide to bind to aPD-L3 OR VISTA binding partner by one of the methods described above fordetermining direct binding. The cell-free assays of the presentinvention are amenable to use of both soluble and/or membrane-boundforms of polypeptides (e.g., PD-L3 OR VISTA polypeptides or biologicallyactive portions thereof, or binding partners to which PD-L3 OR VISTAbinds). In the case of cell-free assays in which a membrane-bound form apolypeptide is used (e.g., a cell-surface PD-L3 OR VISTA), it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of the polypeptide is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucarnide, Triton® X-100,Triton® X-114, Thesit, Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl.dbd.N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either PD-L3 OR VISTA orits binding partner to facilitate separation of complexed fromuncomplexed forms of one or both of the polypeptides, as well as toaccommodate automation of the assay. Binding of a test compound to aPD-L3 OR VISTA polypeptide, or interaction of a PD-L3 OR VISTApolypeptide with its binding partner in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the polypeptides to be bound to a matrix. For example,glutathione-S-transferase/PD-L3 OR VISTA fusion proteins orglutathione-S-transferase/binding partner fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed binding partner polypeptide or PD-L3 OR VISTA polypeptide,and the mixture incubated under conditions conducive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtitre plate wells are washed to remove anyunbound components, the matrix is immobilized in the case of beads, andcomplex formation is determined either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of PD-L3 OR VISTA binding oractivity determined using standard techniques. Other techniques forimmobilizing polypeptides on matrices can also be used in the screeningassays of the invention. In an alternative embodiment, determining theability of the test compound to modulate the activity of a PD-L3 ORVISTA polypeptide can be accomplished by determining the ability of thetest compound to modulate the activity of a molecule that functionsdownstream of PD-L3 OR VISTA, e.g., by interacting with the cytoplasmicdomain of a PD-L3 OR VISTA binding partner. For example, levels ofsecond messengers, the activity of the interacting molecule on anappropriate target, or the binding of the interactor to an appropriatetarget can be determined as previously described.

In another embodiment, modulators of PD-L3 OR VISTA expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of PD-L3 OR VISTA mRNA or polypeptide in thecell is determined. The level of expression of PD-L3 OR VISTA mRNA orpolypeptide in the presence of the candidate compound is compared to thelevel of expression of PD-L3 OR VISTA mRNA or polypeptide in the absenceof the candidate compound. The candidate compound can then be identifiedas a modulator of PD-L3 OR VISTA expression based on this comparison ifthe change is statistically significant.

In yet another aspect of the invention, the PD-L3 OR VISTA polypeptidescan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other polypeptides whichbind to or interact with PD-L3 OR VISTA (“PD-L3 OR VISTA-bindingproteins”, “PD-L3 OR VISTA binding partners”, or “PD-L3 OR VISTA-bp”)and are involved in PD-L3 OR VISTA activity. Such PD-L3 OR VISTA-bindingproteins are also likely to be involved in the propagation of signals bythe PD-L3 OR VISTA polypeptides or PD-L3 OR VISTA targets as, forexample, downstream elements of a PD-L3 OR VISTA-mediated signalingpathway. Alternatively, such PD-L3 OR VISTA-binding polypeptides may bePD-L3 OR VISTA inhibitors. The two-hybrid system is based on the modularnature of most transcription factors, which consist of separableDNA-binding and activation domains. Briefly, the assay utilizes twodifferent DNA constructs. In one construct, the gene that codes for aPD-L3 OR VISTA polypeptide is fused to a gene encoding the DNA bindingdomain of a known transcription factor (e.g, GAL-4). In the otherconstruct, a DNA sequence, from a library of DNA sequences, that encodesan unidentified polypeptide (“prey” or “sample”) is fused to a gene thatcodes for the activation domain of the known transcription factor. Ifthe “bait” and the “prey” polypeptides are able to interact, in vivo,forming a PD-L3 OR VISTA-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g,LacZ) which is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the polypeptide which interacts with the PD-L3 OR VISTApolypeptide.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell-free assay, and the abilityof the agent to modulate the activity of a PD-L3 OR VISTA polypeptidecan be confirmed in vivo, e.g., in an animal such as an animal model forcellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a PD-L3 OR VISTA modulating agent, an antisensePD-L3 OR VISTA nucleic acid molecule, a PD-L3 OR VISTA-specificantibody, or a PD-L3 OR VISTA binding partner) can be used in an animalmodel to determine the efficacy, toxicity, or side effects of treatmentwith such an agent. Alternatively, an agent identified as describedherein can be used in an animal model to determine the mechanism ofaction of such an agent. Furthermore, this invention pertains to uses ofnovel agents identified by the above-described screening assays fortreatments as described herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the PD-L3 OR VISTA nucleotide sequences,described herein, can be used to map the location of the PD-L3 OR VISTAgenes on a chromosome. The mapping of the PD-L3 OR VISTA sequences tochromosomes is an important first step in correlating these sequenceswith genes associated with disease. Briefly, PD-L3 OR VISTA genes can bemapped to chromosomes by preparing PCR primers (preferably 15-25 bp inlength) from the PD-L3 OR VISTA nucleotide sequences. Computer analysisof the PD-L3 OR VISTA sequences can be used to predict primers that donot span more than one exon in the genomic DNA, thus complicating theamplification process. These primers can then be used for PCR screeningof somatic cell hybrids containing individual human chromosomes. Onlythose hybrids containing the human gene corresponding to the PD-L3 ORVISTA sequences will yield an amplified fragment. Somatic cell hybridsare prepared by fusing somatic cells from different mammals (e.g., humanand mouse cells). As hybrids of human and mouse cells grow and divide,they gradually lose human chromosomes in random order, but retain themouse chromosomes. By using media in which mouse cells cannot grow,because they lack a particular enzyme, but human cells can, the onehuman chromosome that contains the gene encoding the needed enzyme willbe retained. By using various media, panels of hybrid cell lines can beestablished. Each cell line in a panel contains either a single humanchromosome or a small number of human chromosomes, and a full set ofmouse chromosomes, allowing easy mapping of individual genes to specifichuman chromosomes (D'Eustachio, P. et al. (1983) Science 220:919-924).Somatic cell hybrids containing only fragments of human chromosomes canalso be produced by using human chromosomes with translocations anddeletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the PD-L3OR VISTA nucleotide sequences to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa PD-L3 OR VISTA sequence to its chromosome include in situhybridization (described in Fan, Y. et al. (1990) Proc Natl. Acad. Sci.USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes, andpre-selection by hybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results in a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofbasic Techniques (Pergamon Press, New York 1988). Reagents forchromosome mapping can be used individually to mark a single chromosomeor a single site on that chromosome, or panels of reagents can be usedfor marking multiple sites and/or multiple chromosomes. Reagentscorresponding to noncoding regions of the genes actually are preferredfor mapping purposes. Coding sequences are more likely to be conservedwithin gene families, thus increasing the chance of cross hybridizationduring chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data Ultimately, complete sequencing of genes fromseveral individuals can be performed to confirm the presence of amutation and to distinguish mutations from polymorphisms. 2. TissueTyping

The PD-L3 OR VISTA sequences of the present invention can also be usedto identify individuals from minute biological samples. Furthermore, thesequences of the present invention can be used to provide an alternativetechnique which determines the actual base-by-base DNA sequence ofselected portions of an individual's genome. Thus, the PD-L3 OR VISTAnucleotide sequences described herein can be used to prepare two PCRprimers from the 5′ and 3′ ends of the sequences. These primers can thenbe used to amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The PD-L3 OR VISTA nucleotide sequences of the invention uniquelyrepresent portions of the human genome. Allelic variation occurs to somedegree in the coding regions of these sequences, and to a greater degreein the noncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO: 1 or 4can comfortably provide positive individual identification with a panelof perhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO: 3 or 6 are used, a more appropriate number of primers forpositive individual identification would be 500-2000.

If a panel of reagents from PD-L3 OR VISTA nucleotide sequencesdescribed herein is used to generate a unique identification databasefor an individual, those same reagents can later be used to identifytissue from that individual. Using the unique identification database,positive identification of the individual, living or dead, can be madefrom extremely small tissue samples.

3. Use of PD-L3 OR VISTA Sequences in Forensic Biology DNA-basedidentification techniques can also be used in forensic biology. Thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i. e., another DNA sequence that is unique to a particularindividual). As mentioned above, actual base sequence information can beused for identification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SEQ ID NO: 1 or 3 are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the PD-L3 OR VISTAnucleotide sequences or portions thereof, e.g., fragments derived fromthe noncoding regions of SEQ ID NO: 1 or 3 having a length of at least20 bases, preferably at least 30 bases. The PD-L3 OR VISTA nucleotidesequences described herein can further be used to provide polynucleotidereagents, e.g., labeled or labelable probes which can be used in, forexample, an in situ hybridization technique, to identify a specifictissue, e.g., lymphocytes. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such PD-L3 OR VISTA probes can be used to identify tissue byspecies and/or by organ type. In a similar fashion, these reagents,e.g., PD-L3 OR VISTA primers or probes can be used to screen tissueculture for contamination (i.e., screen for the presence of a mixture ofdifferent types of cells in a culture).

C Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining PD-L3 OR VISTApolypeptide and/or nucleic acid expression as well as PD-L3 OR VISTAactivity, in the context of a biological sample (e.g., blood, serum,cells, or tissue) to thereby determine whether an individual isafflicted with a disease or disorder, or is at risk of developing adisorder, associated with aberrant or unwanted PD-L3 OR VISTA expressionor activity. The invention also provides for prognostic (or predictive)assays for determining whether an individual is at risk of developing adisorder associated with PD-L3 OR VISTA polypeptide, nucleic acidexpression or activity. For example, mutations in a PD-L3 OR VISTA genecan be assayed in a biological sample. Such assays can be used forprognostic or predictive purpose to thereby prophylactically treat anindividual prior to the onset of a disorder characterized by orassociated with PD-L3 OR VISTA polypeptide, nucleic acid expression oractivity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of PD-L3OR VISTA in clinical trials. These and other agents are described infurther detail in the following sections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of PD-L3 ORVISTA polypeptide or nucleic acid in a biological sample involvesobtaining a biological sample from a test subject and contacting thebiological sample with a compound or an agent capable of detecting PD-L3OR VISTA polypeptide or nucleic acid (e.g., mRNA or genomic DNA) thatencodes PD-L3 OR VISTA polypeptide such that the presence of PD-L3 ORVISTA polypeptide or nucleic acid is detected in the biological sample.A preferred agent for detecting PD-L3 OR VISTA mRNA or genomic DNA is alabeled nucleic acid probe capable of hybridizing to PD-L3 OR VISTA mRNAor genomic DNA. The nucleic acid probe can be, for example, the PD-L3 ORVISTA nucleic acid set forth in SEQ ID NO: 1, or 3, or a portionthereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or500 nucleotides in length and sufficient to specifically hybridize understringent conditions to PD-L3 OR VISTA mRNA or genomic DNA. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein. A preferred agent for detecting PD-L3 OR VISTApolypeptide is an antibody capable of binding to PD-L3 OR VISTApolypeptide, preferably an antibody with a detectable label. Antibodiescan be polyclonal, or more preferably, monoclonal. An intact antibody,or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term“labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i. e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject. That is, the detection method ofthe invention can be used to detect PD-L3 OR VISTA mRNA, polypeptide, orgenomic DNA in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of PD-L2 mRNA includeNorthern hybridizations and in situ hybridizations. In vitro techniquesfor detection of PD-L3 OR VISTA polypeptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of PD-L3 OR VISTAgenomic DNA include Southern hybridizations. Furthermore, in vivotechniques for detection of PD-L3 OR VISTA polypeptide includeintroducing into a subject a labeled anti-PD-L3 OR VISTA antibody. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. In one embodiment, the biological sample containspolypeptide molecules from the test subject. Alternatively, thebiological sample can contain mRNA molecules from the test subject orgenomic DNA molecules from the test subject. A preferred biologicalsample is a serum sample isolated by conventional means from a subject.In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting PD-L3 OR VISTApolypeptide, mRNA, or genomic DNA, such that the presence of PD-L3 ORVISTA polypeptide, mRNA or genomic DNA is detected in the biologicalsample, and comparing the presence of PD-L3 OR VISTA polypeptide, mRNAor genomic DNA in the control sample with the presence of PD-L3 OR VISTApolypeptide, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of PD-L3OR VISTA in a biological sample. For example, the kit can comprise alabeled compound or agent capable of detecting PD-L3 OR VISTApolypeptide or mRNA in a biological sample; means for determining theamount of PD-L3 OR VISTA in the sample; and means for comparing theamount of PD-L3 OR VISTA in the sample with a standard. The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect PD-L3 OR VISTApolypeptide or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted PD-L3 OR VISTA expression oractivity. As used herein, the term “aberrant” includes a PD-L3 OR VISTAexpression or activity which deviates from the wild type PD-L3 OR VISTAexpression or activity. Aberrant expression or activity includesincreased or decreased expression or activity, as well as expression oractivity which does not follow the wild type developmental pattern ofexpression or the subcellular pattern of expression. For example,aberrant PD-L3 OR VISTA expression or activity is intended to includethe cases in which a mutation in the PD-L3 OR VISTA gene causes thePD-L3 OR VISTA gene to be under-expressed or over-expressed andsituations in which such mutations result in a non-functional PD-L3 ORVISTA polypeptide or a polypeptide which does not function in awild-type fashion, e.g., a polypeptide which does not interact with aPD-L3 OR VISTA binding partner, or one which interacts with a non-PD-L3OR VISTA binding partner. As used herein, the term “unwanted” includesan unwanted phenomenon involved in a biological response such as immunecell activation. For example, the term unwanted includes a PD-L3 ORVISTA expression or activity which is undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in PD-L3OR VISTA polypeptide activity or nucleic acid expression, such as anautoimmune disorder, an immunodeficiency disorder, an immune systemdisorder such as autoimmunity, allergic or inflammatory disorder orcancer. Thus, the present invention provides a method for identifying adisease or disorder associated with aberrant or unwanted PD-L3 OR VISTAexpression or activity in which a test sample is obtained from a subjectand PD-L3 OR VISTA polypeptide or nucleic acid (e.g., mRNA or genomicDNA) is detected, wherein the presence of PD-L3 OR VISTA polypeptide ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant or unwanted PD-L3 ORVISTA expression or activity. As used herein, a “test sample” refers toa biological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., cerebrospinal fluid orserum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted PD-L3 OR VISTA expression oractivity. For example, such methods can be used to determine whether asubject can be effectively treated with an agent for an autoimmunedisorder, immunodeficiency disorder, immune system cancer, or allergicor inflammatory disorder. Thus, the present invention provides methodsfor determining whether a subject can be effectively treated with anagent for a disorder associated with aberrant or unwanted PD-L3 OR VISTAexpression or activity in which a test sample is obtained and PD-L3 ORVISTA polypeptide or nucleic acid expression or activity is detected(e.g., wherein the abundance of PD-L3 OR VISTA polypeptide or nucleicacid expression or activity is diagnostic for a subject that can beadministered the agent to treat a disorder associated with aberrant orunwanted PD-L3 OR VISTA expression or activity). The methods of theinvention can also be used to detect genetic alterations in a PD-L3 ORVISTA gene, thereby determining if a subject with the altered gene is atrisk for a disorder characterized by misregulation in PD-L3 OR VISTApolypeptide activity or nucleic acid expression, such as an autoimmunedisorder, an immunodeficiency disorder, an immune system cancer, anallergic disorder, or an inflammatory disorder. The methods describedherein may be performed, for example, by utilizing pre-packageddiagnostic kits comprising at least one probe nucleic acid or antibodyreagent described herein, which may be conveniently used, e.g., inclinical settings to diagnose patients exhibiting symptoms or familyhistory of a disease or illness involving a PD-L3 OR VISTA gene.Furthermore, any cell type or tissue in which PD-L3 OR VISTA isexpressed may be utilized in the prognostic assays described herein.

3. Monitoring of Effects During Clinical Trials Monitoring the influenceof agents (e.g., drugs) on the expression or activity of a PD-L3 ORVISTA polypeptide (e.g., the modulation of cell proliferation and/ormigration) can be applied not only in basic drug screening, but also inclinical trials. For example, the effectiveness of an agent determinedby a screening assay as described herein to increase PD-L3 OR VISTA geneexpression, polypeptide levels, or upregulate PD-L3 OR VISTA activity,can be monitored in clinical trials of subjects exhibiting decreasedPD-L3 OR VISTA gene expression, polypeptide levels, or downregulatedPD-L3 OR VISTA activity. Alternatively, the effectiveness of an agentdetermined by a screening assay to decrease PD-L3 OR VISTA geneexpression, polypeptide levels, or downregulate PD-L3 OR VISTA activity,can be monitored in clinical trials of subjects exhibiting increasedPD-L3 OR VISTA gene expression, polypeptide levels, or PD-L3 OR VISTAactivity. As noted PD-L3 OR VISTA is expressed on many hematopoieticcell types including APCs (macrophages and myeloid dendritic cells), andCD4+ T cells, and more specifically is expressed on CD11c⁺ DCs, CD4⁺ Tcells (including both Foxp3⁻ effector T cells and Foxp3⁺ nTregs), CD8⁺ Tcells, and Gr1⁺ granulocytes, and expressed at low levels on B cells andNK cells In such clinical trials, the expression or activity of a PD-L3OR VISTA gene, and preferably, other genes that have been implicated in,for example, a PD-L3 OR VISTA-associated disorder can be used as a “readout” or marker of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including PD-L3 ORVISTA, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates PD-L3 OR VISTAactivity (e.g., identified in a screening assay as described herein) canbe identified. Thus, to study the effect of agents on PD-L3 ORVISTA-associated disorders, for example, in a clinical trial, cells canbe isolated and RNA prepared and analyzed for the levels of expressionof PD-L3 OR VISTA and other genes implicated in the PD-L3 ORVISTA-associated disorder, respectively. The levels of gene expression(e.g., a gene expression pattern) can be quantified by Northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of polypeptide produced, by one of the methods as describedherein, or by measuring the levels of activity of PD-L3 OR VISTA orother genes. In this way, the gene expression pattern can serve as amarker, indicative of the physiological response of the cells to theagent. Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent. In apreferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide,nucleic acid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a PD-L3 OR VISTApolypeptide, mRNA, or genomic DNA in the preadministration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the PD-L3 OR VISTApolypeptide, mRNA, or genomic DNA in the post-administration samples;(v) comparing the level of expression or activity of the PD-L3, OR VISTApolypeptide, mRNA, or genomic DNA in the pre-administration sample withthe PD-L3 OR VISTA polypeptide, mRNA, or genomic DNA in the postadministration sample or samples; and (vi) altering the administrationof the agent to the subject accordingly. For example, increasedadministration of the agent may be desirable to increase the expressionor activity of PD-L3 OR VISTA to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of PD-L3 OR VISTA to lower levels than detected, i.e., todecrease the effectiveness of the agent. According to such anembodiment, PD-L3 OR VISTA expression or activity may be used as anindicator of the effectiveness of an agent, even in the absence of anobservable phenotypic response.

D. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disordercharacterized by insufficient or excessive production of PD-L3 OR VISTAprotein or production of PD-L3 OR VISTA protein forms which havedecreased or aberrant activity compared to PD-L3 OR VISTA wild typeprotein. Moreover, the anti-PD-L3 OR VISTA antibodies of the inventioncan be used to detect and isolate PD-L3 OR VISTA proteins, regulate thebioavailability of PD-L3 OR VISTA proteins, and modulate PD-L3 OR VISTAactivity e.g., by modulating the interaction of PD-L3 OR VISTA with itscounter receptor.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedPD-L3 OR VISTA expression or activity, by administering to the subject aPD-L3 OR VISTA polypeptide or an agent which modulates PD-L3 OR VISTAexpression or at least one PD-L3 OR VISTA activity. Subjects at risk fora disease or disorder which is caused or contributed to by aberrant orunwanted PD-L3 OR VISTA expression or activity can be identified by, forexample, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the PD-L3 OR VISTAaberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofPD-L3 OR VISTA aberrancy, for example, a PD-L3 OR VISTA polypeptide,PD-L3 OR VISTA agonist or PD-L3 OR VISTA antagonist (e.g., an anti-PD-L3OR VISTA antibody) agent can be used for treating the subject. Theappropriate agent can be determined based on screening assays describedherein.

2. Therapeutic Methods

An important aspect of the invention pertains to methods of modulatingPD-L3 OR VISTA expression or activity or interaction with its naturalbinding partners, Relevant to therapy PD-L3 OR VISTA has beendemonstrated to inhibit CD28 costimulation, to inhibit TCR activation ofimmune cells, to inhibit proliferation of activated immune cells (CD4+and CD8+ T cells), to inhibit cytokine production by T cells (IL-2,gamma interferon) and to transmit an inhibitory signal to immune cells.Accordingly, the activity and/or expression of PD-L3 OR VISTA, as wellas the interaction between PD-L3 OR VISTA and its binding partner)s) onT cells can be modulated in order to modulate the immune response.Because PD-L3 OR VISTA binds to inhibitory receptors (on T cells),upregulation of PD-L3 OR VISTA activity should result in downregulationof immune responses, whereas downregulation of PD-L3 OR VISTA activityshould results in upregulation of immune responses. In a preferredembodiment, PD-L3 OR VISTA binds to inhibitory receptors. As notedpreviously, counterintuitively PD-L3 OR VISTA specific antibodiesproduced by Applicant which in vitro (in the presence of PD-L3 ORVISTA-Ig) enhance the suppressive activities of PD-L3 OR VISTA-Ig fusionproteins (i.e., these antibodies enhance the suppression of PD-L3 ORVISTA related activities such as effects of PD-L3 OR VISTA on cytokineproduction, T cell proliferation, differentiation or activation andother functions noted previously), behave oppositely to what would beexpected in vivo, i.e., these antibodies have been found to beimmunosuppressive in vivo.

Modulatory methods of the invention involve contacting a cell with aPD-L3 OR VISTA polypeptide or agent that modulates one or more of theactivities of PD-L3 OR VISTA polypeptide activity associated with thecell, e.g., an agent that modulates expression or activity of PD-L3 ORVISTA and/or modulates the interaction of PD-L3 OR VISTA and its naturalbinding partner(s). An agent that modulates PD-L3 OR VISTA polypeptideactivity can be an agent as described herein, such as a nucleic acid ora polypeptide, a naturally-occurring binding partner of a PD-L3 OR VISTApolypeptide a PD-L3 OR VISTA antibody, a PD-L3 OR VISTA agonist orantagonist, a peptidomimetic of a PD-L3 OR VISTA agonist or antagonist,a PD-L3 OR VISTA peptidomimetic, or other small molecule. Soluble formsof PD-L3 OR VISTA may also be used to interfere with the binding ofPD-L3 OR VISTA to any of its natural binding partner(s) or ligands.

An agent that modulates the expression of PD-L3 OR VISTA is, e.g., anantisense nucleic acid molecule, triplex oligonucleotide, ribozyme, orrecombinant vector for expression of a PD-L3 OR VISTA polypeptide. Forexample, an oligonucleotide complementary to the area around a PD-L3 ORVISTA polypeptide translation initiation site can be synthesized. One ormore antisense oligonucleotides can be added to cell media, typically at200 mug/ml, or administered to a patient to prevent the synthesis of aPD-L3 OR VISTA polypeptide. The antisense oligonucleotide is taken up bycells and hybridizes to a PD-L3 OR VISTA mRNA to prevent translation.Alternatively, an oligonucleotide which binds double-stranded DNA toform a triplex construct to prevent DNA unwinding and transcription canbe used. As a result of either, synthesis of PD-L3 OR VISTA polypeptideis blocked. When PD-L3 OR VISTA expression is modulated, preferably,such modulation occurs by a means other than by knocking out the PD-L3OR VISTA gene.

Agents which modulate expression, by virtue of the fact that theycontrol the amount of PD-L3 OR VISTA in a cell, also modulate the totalamount of PD-L3 OR VISTA activity in a cell. In one embodiment, theagent the modulates PD-L3 OR VISTA stimulates one or more PD-L3 OR VISTAactivities. Examples of such stimulatory agents include active PD-L3 ORVISTA polypeptide and a nucleic acid molecule encoding PD-L3 OR VISTAthat has been introduced into the cell. In another embodiment, the agentinhibits one or more PD-L3 OR VISTA activities. Examples of suchinhibitory agents include antisense PD-L3 OR VISTA nucleic acidmolecules, anti-PD-L3 OR VISTA antibodies, PD-L3 OR VISTA inhibitors,and compounds identified in the subject screening assays. In a furtherpreferred embodiment, an inhibitory agent is a combination of ananti-PD-L3 OR VISTA antibody and an anti-PD-L1 or anti-PD-L2 antibody.These modulatory methods can be performed in vitro (e.g., by contactingthe cell with the agent) or, alternatively, by contacting an agent withcells in vivo (e.g., by administering the agent to a subject). As such,the present invention provides methods of treating an individualafflicted with a condition or disorder that would benefit from up- ordown-modulation of a PD-L3 OR VISTA polypeptide, e.g., a disordercharacterized by unwanted, insufficient, or aberrant expression oractivity of a PD-L3 OR VISTA polypeptide or nucleic acid molecule. Inone embodiment, the method involves administering an agent (e.g., anagent identified by a screening assay described herein), or combinationof agents that modulates (e.g., upregulates or downregulates) PD-L3 ORVISTA expression or activity. In another embodiment, the method involvesadministering a PD-L3 OR VISTA polypeptide or nucleic acid molecule astherapy to compensate for reduced, aberrant, or unwanted PD-L3 OR VISTAexpression or activity.

Diseases treatable with the subject PD-L3 OR VISTA binding agents areidentified previously and include various inflammatory, autoimmune,cancer, allergic and infectious disorders. A particularly preferredindication is multiple sclerosis.

Stimulation of PD-L3 OR VISTA activity is desirable in situations inwhich PD-L3 OR VISTA is abnormally downregulated and/or in whichincreased PD-L3 OR VISTA activity is likely to have a beneficial effect.Likewise, inhibition of PD-L3 OR VISTA activity is desirable insituations in which PD-L3 OR VISTA is abnormally upregulated and/or inwhich decreased PD-L3 OR VISTA activity is likely to have a beneficialeffect. Exemplary agents for use in downmodulating PD-L3 OR VISTA (i.e.,PD-L3 OR VISTA antagonists) include, e.g., antisense nucleic acidmolecules, antibodies that recognize and block PD-L3 OR VISTA,combinations of antibodies that recognize and block PD-L3 OR VISTA andantibodies that recognize and block PD-L3 OR VISTA counter receptors,and compounds that block the interaction of PD-L3 OR VISTA with itsnaturally occurring binding partner(s) on an immune cell (e.g., soluble,monovalent PD-L3 OR VISTA molecules; soluble forms of PD-L3 OR VISTAmolecules that do not bind Fc receptors on antigen presenting cells;soluble forms of PD-L3 OR VISTA binding partners; and compoundsidentified in the subject screening assays). Exemplary agents for use inupmodulating PD-L3 OR VISTA (i.e., PD-L3 OR VISTA agonists) include,e.g., nucleic acid molecules encoding PD-L3 OR VISTA polypeptides,multivalent forms of PD-L3 OR VISTA, compounds that increase theexpression of PD-L3 OR VISTA, compounds that enhance the interaction ofPD-L3 OR VISTA with its naturally occurring binding partners and cellsthat express PD-L3 OR VISTA.

3. Downregulation of Immune Responses

There are numerous embodiments of the invention for upregulating theinhibitory function of a PD-L3 OR VISTA polypeptide to therebydownregulate immune responses. Downregulation can be in the form ofinhibiting or blocking an immune response already in progress, or mayinvolve preventing the induction of an immune response. The functions ofactivated immune cells can be inhibited by downregulating immune cellresponses or by inducing specific anergy in immune cells, or both. Forexample, in embodiments where PD-L3 OR VISTA binds to an inhibitoryreceptor, forms of PD-L3 OR VISTA that bind to the inhibitory receptor,e.g., multivalent PD-L3 OR VISTA on a cell surface, can be used todownmodulate the immune response. In one embodiment of the invention, anactivating antibody used to stimulate PD-L3 OR VISTA activity is abispecific antibody. For example, such an antibody can comprise a PD-L3OR VISTA binding site and another binding site which targets a cellsurface receptor on an immune cell, e.g., a T cell, a B cell, or amyeloid cell. In one embodiment, such an antibody, in addition tocomprising a PD-L3 OR VISTA binding site, can further comprise a bindingsite which binds to a B cell antigen receptor, a T cell antigenreceptor, or an Fc receptor, in order to target the molecule to aspecific cell population. Selection of this second antigen for thebispecific antibody provides flexibility in selection of cell populationto be targeted for inhibition. Agents that promote a PD-L3 OR VISTAactivity or which enhance the interaction of PD-L3 OR VISTA with itsnatural binding partners (e.g., PD-L3 OR VISTA activating antibodies orPD-L3 OR VISTA activating small molecules) can be identified by theirability to inhibit immune cell proliferation and/or effector function,or to induce anergy when added to an in vitro assay. For example, cellscan be cultured in the presence of an agent that stimulates signaltransduction via an activating receptor. A number of art-recognizedreadouts of cell activation can be employed to measure, e.g., cellproliferation or effector function (e.g., antibody production, cytokineproduction, phagocytosis) in the presence of the activating agent. Theability of a test agent to block this activation can be readilydetermined by measuring the ability of the agent to effect a decrease inproliferation or effector function being measured. In one embodiment, atlow antigen concentrations, PD-L3 OR VISTA immune cell interactionsinhibit strong B7-CD28 signals. In another embodiment, at high antigenconcentrations, PD-L3 OR VISTA immune cell interactions may reducecytokine production but not inhibit T cell proliferation. Accordingly,the ability of a test compound to block activation can be determined bymeasuring cytokine production and/or proliferation at differentconcentrations of antigen.

In one embodiment of the invention, tolerance is induced againstspecific antigens by co-administering an antigen with a PD-L3 OR VISTAagonist. For example, tolerance can be induced to specific polypeptides.In one embodiment, immune responses to allergens or foreign polypeptidesto which an immune response is undesirable can be inhibited. Forexample, patients that receive Factor VIII frequently generateantibodies against this clotting factor. Co-administration of an agentthat stimulates PD-L3 OR VISTA activity or interaction with its naturalbinding partner, with recombinant factor VIII (or physically linkingPD-L3 OR VISTA to Factor VIII, e.g., by cross-linking) can result inimmune response downmodulation.

In one embodiment, a PD-L3 OR VISTA agonist and another agent that canblock activity of costimulatory receptors on an immune cell can be usedto downmodulate immune responses. Exemplary molecules include: agonistsforms of other PD ligands, soluble forms of CTLA-4, anti-B7-1antibodies, anti-B7-2 antibodies, or combinations thereof.Alternatively, two separate peptides (for example, a PD-L3 OR VISTApolypeptide with blocking forms of B7-2 and/or B7-1 polypeptides), or acombination of antibodies (e.g., activating antibodies against a PD-L3OR VISTA polypeptide with blocking anti-B7-2 and/or anti-B7-1 monoclonalantibodies) can be combined as a single composition or administeredseparately (simultaneously or sequentially) to downregulate immune cellmediated immune responses in a subject. Furthermore, a therapeuticallyactive amount of one or more peptides having a PD-L3 OR VISTApolypeptide activity, along with one or more polypeptides having B7-1and/or B7-1 activity, can be used in conjunction with otherdownmodulating reagents to influence immune responses. Examples of otherimmunomodulating reagents include antibodies that block a costimulatorysignal (e.g., against CD28 or ICOS), antibodies that activate aninhibitory signal via CTLA4, and/or antibodies against other immune cellmarkers (e.g., against CD40, CD40 ligand, or cytokines), fusion proteins(e.g., CTLA4-Fc or PD-1-Fc), and immunosuppressive drugs (e.g.,rapamycin, cyclosporine A, or FK506). The PD-L3 OR VISTA polypeptidesmay also be useful in the construction of therapeutic agents which blockimmune cell function by destruction of cells. For example, portions of aPD-L3 OR VISTA polypeptide can be linked to a toxin to make a cytotoxicagent capable of triggering the destruction of cells to which it binds.

For making cytotoxic agents, polypeptides of the invention may belinked, or operatively attached, to toxins using techniques that areknown in the art. A wide variety of toxins are known that may beconjugated to polypeptides or antibodies of the invention. Examplesinclude: numerous useful plant-, fungus- or even bacteria-derivedtoxins, which, by way of example, include: various A chain toxins,particularly ricin A chain; ribosome inactivating proteins such assaporin or gelonin; alpha-sarcin; aspergillin; restrictocin; andribonucleases such as placental ribonuclease, angiogenic, diphtheriatoxin, or pseudomonas exotoxin. A preferred toxin moiety for use inconnection with the invention is toxin A chain which has been treated tomodify or remove carbohydrate residues, deglycosylated A chain. (U.S.Pat. No. 5,776,427).

Infusion of one or a combination of such cytotoxic agents (e.g., PD-L3OR VISTA ricin (alone or in combination with PD-L1-ricin), into apatient may result in the death of immune cells, particularly in lightof the fact that activated immune cells that express higher amounts ofPD-L3 OR VISTA binding partners, . For example, because PD-1 is inducedon the surface of activated lymphocytes, a PD-L3 OR VISTA polypeptidecan be used to target the depletion of these specific cells by Fc-Rdependent mechanisms or by ablation by conjugating a cytotoxic drug(e.g., ricin, saporin, or calicheamicin) to the PD-L3 OR VISTApolypeptide. In one another embodiment, the toxin can be conjugated toan anti-PD-L3 OR VISTA antibody in order to target for death PD-L3 ORVISTA-expressing antigen-presenting cell. In a further embodiment, thePD-L3 OR VISTA-antibody-toxin can be a bispecific antibody. Suchbispecific antibodies are useful for targeting a specific cellpopulation, e.g., using a marker found only on a certain type of cell,e.g., B lymphocytes, monocytes, dendritic cells, or Langerhans cells.Downregulating immune responses by activating PD-L3 OR VISTA activity orthe PD-L3 OR VISTA-immune cell interaction (and thus stimulating thenegative signaling function of PD-L3 OR VISTA) is useful indownmodulating the immune response, e.g., in situations of tissue, skinand organ transplantation, in graft-versus-host disease (GVHD), orallergies, or in autoimmune diseases such as systemic lupuserythematosus and multiple sclerosis. For example, blockage of immunecell function results in reduced tissue destruction in tissuetransplantation. Typically, in tissue transplants, rejection of thetransplant is initiated through its recognition as foreign by immunecells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which promotes the activity of PD-L3 ORVISTA or the interaction of PD-L3 OR VISTA with its natural bindingpartner(s), on immune cells (such as a soluble, multimeric form of aPD-L3 OR VISTA polypeptide) alone or in conjunction with anotherdownmodulatory agent prior to or at the time of transplantation caninhibit the generation of a costimulatory signal. Moreover, promotion ofPD-L3 OR VISTA activity may also be sufficient to anergize the immunecells, thereby inducing tolerance in a subject.

To achieve sufficient immunosuppression or tolerance in a subject, itmay also be desirable to block the costimulatory function of othermolecules. For example, it may be desirable to block the function ofB7-1 and B7-2 by administering a soluble form of a combination ofpeptides having an activity of each of these antigens or blockingantibodies against these antigens (separately or together in a singlecomposition) prior to or at the time of transplantation. Alternatively,it may be desirable to promote inhibitory activity of PD-L3 OR VISTA andinhibit a costimulatory activity of B7-1 and/or B7-2. Otherdownmodulatory agents that can be used in connection with thedownmodulatory methods of the invention include, for example, agentsthat transmit an inhibitory signal via CTLA4, soluble forms of CTLA4,antibodies that activate an inhibitory signal via CTLA4, blockingantibodies against other immune cell markers, or soluble forms of otherreceptor ligand pairs (e.g., agents that disrupt the interaction betweenCD40 and CD40 ligand (e.g., anti CD40 ligand antibodies)), antibodiesagainst cytokines, or immunosuppressive drugs. For example, activatingPD-L3 OR VISTA activity or the interaction of PD-L3 OR VISTA with itsnatural binding partner(s), is useful in treating autoimmune disease.Many autoimmune disorders are the result of inappropriate activation ofimmune cells that are reactive against self tissue and which promote theproduction of cytokines and autoantibodies involved in the pathology ofthe diseases. Preventing the activation of autoreactive immune cells mayreduce or eliminate disease symptoms. Administration of agents thatpromote activity of PD-L3 OR VISTA or PD-L3 OR VISTA interaction withits natural binding partner(s), may induce antigen-specific tolerance ofautoreactive immune cells which could lead to long-term relief from thedisease. Additionally, co-administration of agents which blockcostimulation of immune cells by disrupting receptor-ligand interactionsof B7 molecules with costimulatory receptors may be useful in inhibitingimmune cell activation to prevent production of autoantibodies orcytokines which may be involved in the disease process. The efficacy ofreagents in preventing or alleviating autoimmune disorders can bedetermined using a number of well-characterized animal models of humanautoimmune diseases. Examples include murine experimental autoimmuneencephalitis, systemic lupus erythematosus in MRL/lpr/lpr mice or NZBhybrid mice, murine autoimmune collagen arthritis, diabetes mellitus inNOD mice and BB rats, and murine experimental myasthenia gravis (seePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.840-856).

Inhibition of immune cell activation is useful therapeutically in thetreatment of allergies and allergic reactions, e.g., by inhibiting IgEproduction. An agent that promotes PD-L3 OR VISTA activity or PD-L3 ORVISTA interaction with its natural binding partner(s) can beadministered to an allergic subject to inhibit immune cell-mediatedallergic responses in the subject. Stimulation PD-L3 OR VISTA activityor interaction with its natural binding partner(s), can be accompaniedby exposure to allergen in conjunction with appropriate MHC molecules.Allergic reactions can be systemic or local in nature, depending on theroute of entry of the allergen and the pattern of deposition of IgE onmast cells or basophils. Thus, immune cell-mediated allergic responsescan be inhibited locally or systemically by administration of an agentthat promotes PD-L3 OR VISTA activity or PD-L3 OR VISTA-immune cellinteractions.

Inhibition of immune cell activation through stimulation of PD-L3 ORVISTA activity or PD-L3 OR VISTA interaction with its natural bindingpartner(s), may also be important therapeutically in pathogenicinfections of immune cells (e.g., by viruses or bacteria). For example,in the acquired immune deficiency syndrome (AIDS), viral replication isstimulated by immune cell activation. Stimulation of PD-L3 OR VISTAactivity may result in inhibition of viral replication and therebyameliorate the course of AIDS.

Downregulation of an immune response via stimulation of PD-L3 OR VISTAactivity or PD-L3 OR VISTA interaction with its natural bindingpartner(s), may also be useful in treating an autoimmune attack ofautologous tissues. Thus, conditions that are caused or exacerbated byautoimmune attack (e.g., heart disease, myocardial infarction oratherosclerosis) may be ameliorated or improved by increasing PD-L3 ORVISTA activity or PD-L3 OR VISTA biding to its natural binding partner.It is therefore within the scope of the invention to modulate conditionsexacerbated by autoimmune attack, such as autoimmune disorders (as wellas conditions such as heart disease, myocardial infarction, andatherosclerosis) by stimulating PD-L3 OR VISTA activity or PD-L3 ORVISTA interaction with its counter receptor.

4. Upregulation of Immune Responses

Inhibition of PD-L3 OR VISTA activity or PD-L3 OR VISTA interaction withits natural binding partner(s), as a means of upregulating immuneresponses is also useful in therapy. Upregulation of immune responsescan be in the form of enhancing an existing immune response or elicitingan initial immune response. For example, enhancing an immune responsethrough inhibition of PD-L3 OR VISTA activity is useful in cases ofinfections with microbes, e.g., bacteria, viruses, or parasites, or incases of immunosuppression. For example, in one embodiment, an agentthat inhibits PD-L3 OR VISTA activity, e.g., a non-activating antibody(i.e., a blocking antibody) against PD-L3 OR VISTA, or a soluble form ofPD-L3 OR VISTA, is therapeutically useful in situations whereupregulation of antibody and cell-mediated responses, resulting in morerapid or thorough clearance of a virus, bacterium, or parasite, would bebeneficial. These conditions include viral skin diseases such as Herpesor shingles, in which case such an agent can be delivered topically tothe skin. In addition, systemic viral diseases such as influenza, thecommon cold, and encephalitis might be alleviated by the administrationof such agents systemically. In certain instances, it may be desirableto further administer other agents that upregulate immune responses, forexample, forms of B7 family members that transduce signals viacostimulatory receptors, in order further augment the immune response.

Alternatively, immune responses can be enhanced in an infected patientby removing immune cells from the patient, contacting immune cells invitro with an agent that inhibits the PD-L3 OR VISTA activity or PD-L3OR VISTA interaction with its natural binding partner(s), andreintroducing the in vitro-stimulated immune cells into the patient. Inanother embodiment, a method of enhancing immune responses involvesisolating infected cells from a patient, e.g., virally infected cells,transfecting them with a nucleic acid molecule encoding a form of PD-L3OR VISTA that cannot bind its natural binding partner(s), such that thecells express all or a portion of the PD-L3 OR VISTA molecule on theirsurface, and reintroducing the transfected cells into the patient. Thetransfected cells may be capable of preventing an inhibitory signal to,and thereby activating, immune cells in vivo.

A agent that inhibits PD-L3 OR VISTA activity or PD-L3 OR VISTAinteraction with its natural binding partner(s), can be usedprophylactically in vaccines against various polypeptides, e.g.,polypeptides derived from pathogens. Immunity against a pathogen, e.g.,a virus, can be induced by vaccinating with a viral polypeptide alongwith an agent that inhibits PD-L3 OR VISTA activity, in an appropriateadjuvant. Alternately, a vector comprising genes which encode for both apathogenic antigen and a form of PD-L3 OR VISTA that blocks PD-L3 ORVISTA interaction with immune cells can be used for vaccination. Nucleicacid vaccines can be administered by a variety of means, for example, byinjection (e.g., intramuscular, intradermal, or the biolistic injectionof DNA-coated gold particles into the epidermis with a gene gun thatuses a particle accelerator or a compressed gas to inject the particlesinto the skin (Haynes et al. (1996) J. Biotechnol. 44:37)).Alternatively, nucleic acid vaccines can be administered by non-invasivemeans. For example, pure or lipid-formulated DNA can be delivered to therespiratory system or targeted elsewhere, e.g., Peyers patches by oraldelivery of DNA (Schubbert (1997) Proc Natl. Acad. Sci. USA 94:961).Attenuated microorganisms can be used for delivery to mucosal surfaces(Sizemore et al. (1995) Science 270:29).

In another embodiment, the antigen in the vaccine is a self-antigen.Such a vaccine is useful in the modulation of tolerance in an organism.Immunization with a self antigen and an agent that blocks PD-L3 OR VISTAactivity or PD-L3 OR VISTA interaction with its natural binding partnercan break tolerance (i.e., interfere with tolerance of a self antigen).Such a vaccine may also include adjuvants such as alum or cytokines(e.g., GM-CSF, IL-12, B7-1, or B7-2). In one embodiment, an agent whichinhibits PD-L3 OR VISTA activity or PD-L3 OR VISTA interaction with itsnatural binding partner(s), can be administered with class I MHCpolypeptides by, for example, a cell transfected to coexpress a PD-L3 ORVISTA polypeptide or blocking antibody and MHC class I .alpha. chainpolypeptide and beta2 microglobulin to result in activation of T cellsand provide immunity from infection. For example, viral pathogens forwhich vaccines are useful include: hepatitis B, hepatitis C,Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malariaand schistosomiasis.

In another application, inhibition of PD-L3 OR VISTA activity or PD-L3OR VISTA interaction with its natural binding partner(s), can be usefulin the treatment of tumor immunity. Tumor cells (e.g., sarcoma,melanoma, lymphoma, leukemia, neuroblastoma, or carcinoma) can betransfected with a nucleic acid molecule that inhibits PD-L3 OR VISTAactivity. These molecules can be, e.g., nucleic acid molecules which areantisense to PD-L3 OR VISTA, or can encode non-activating anti-PD-L3 ORVISTA antibodies. These molecules can also be the variable region of ananti-PD-L3 OR VISTA antibody. If desired, the tumor cells can also betransfected with other polypeptides which activate costimulation (e.g.,B7-1 or B7-2). The transfected tumor cells are returned to the patient,which results in inhibition (e.g., local inhibition) of PD-L3 OR VISTAactivity Alternatively, gene therapy techniques can be used to target atumor cell for transfection in vivo.

Stimulation of an immune response to tumor cells can also be achieved byinhibiting PD-L3 OR VISTA activity or PD-L3 OR VISTA interaction withits natural binding partner(s), by treating a patient with an agent thatinhibits PD-L3 OR VISTA activity or PD-L3 OR VISTA interaction with itsnatural binding partner(s). Preferred examples of such agents include,e.g., antisense nucleic acid molecules, antibodies that recognize andblock PD-L3 OR VISTA, and compounds that block the interaction of PD-L3OR VISTA with its naturally occurring binding partner(s) on an immunecell (e.g., soluble, monovalent PD-L3 OR VISTA molecules; soluble formsof PD-L3 OR VISTA molecules that do not bind to Fc receptors on antigenpresenting cells; soluble forms of PD-L3 OR VISTA binding partner(s);and compounds identified in the subject screening assays). In addition,tumor cells which lack MHC class I or MHC class II molecules, or whichfail to express sufficient amounts of MHC class I or MHC class IImolecules, can be transfected with nucleic acid encoding all or aportion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I .alpha. chain polypeptide and beta2 microglobulin polypeptide oran MHC class II .alpha. chain polypeptide and an MHC class II .beta.chain polypeptide to thereby express MHC class I or MHC class Hpolypeptides on the cell surface. Expression of the appropriate class Ior class II MHC in conjunction with an PD-L3 OR VISTA inhibitingpolypeptide or antisense nucleic acid induces a T cell mediated immuneresponse against the transfected tumor cell. Optionally, a gene encodingan antisense construct which blocks expression of an MHC classII-associated polypeptide, such as the invariant chain, can also becotransfected with a DNA encoding a PD-L3 OR VISTA inhibitingpolypeptide or antisense nucleic acid to promote presentation of tumorassociated antigens and induce tumor specific immunity. Expression ofB7-1 by B7-negative murine tumor cells has been shown to induce T cellmediated specific immunity accompanied by tumor rejection and prolongedprotection to tumor challenge in mice (Chen, L. et al. (1992) Cell71:1093-1102; Townsend, S. E. and Allison, J. P. (1993) Science259:368-370; Baskar, S. et al. (1993) Proc Natl. Acad. Sci.90:5687-5690). Thus, the induction of an immune cell-mediated immuneresponse in a human subject can be sufficient to overcome tumor-specifictolerance in the subject. In another embodiment, the immune response canbe stimulated by the inhibition of PD-L3 OR VISTA activity or PD-L3 ORVISTA interaction with its natural binding partner(s), such thatpreexisting tolerance is overcome. For example, immune responses againstantigens to which a subject cannot mount a significant immune response,e.g., tumor-specific antigens, can be induced by administering an agentthat inhibits the activity of PD-L3 OR VISTA activity or the ability ofPD-L3 OR VISTA to bind to its natural binding partner, can be used asadjuvants to boost responses to foreign antigens in the process ofactive immunization.

In one embodiment, immune cells are obtained from a subject and culturedex vivo in the presence of an agent that that inhibits PD-L3 OR VISTAactivity or PD-L3 OR VISTA interaction with its natural bindingpartner(s), to expand the population of immune cells. In a furtherembodiment the immune cells are then administered to a subject. Immunecells can be stimulated to proliferate in vitro by, for example,providing the immune cells with a primary activation signal and acostimulatory signal, as is known in the art. Various forms of PD-L3 ORVISTA polypeptides or agents that inhibit PD-L3 OR VISTA activity canalso be used to costimulate proliferation of immune cells. In oneembodiment, immune cells are cultured ex vivo according to the methodsdescribed in PCT Application No. WO 94/29436. The costimulatory moleculecan be soluble, attached to a cell membrane or attached to a solidsurface, such as a bead.

In an additional embodiment, in performing any of the methods describedherein, it is within the scope of the invention to upregulate an immuneresponse by administering one or more additional agents. For example,the use of other agents known to stimulate the immune response, such ascytokines, adjuvants, or stimulatory forms of costimulatory molecules ortheir ligands can be used in conjunction with an agent that inhibitsPD-L3 OR VISTA activity or PD-L3 OR VISTA interaction with its naturalbinding partner(s).

E. Identification of Cytokines Modulated by Modulation of PD-L3 OR VISTAActivity or PD-L3 OR VISTA-Interactions with its Counter Receptor on TCells

The PD-L3 OR VISTA molecules described herein can be used to identifycytokines which are produced by or whose production is enhanced orinhibited in immune cells in response to modulation of PD-L3 OR VISTAactivity or PD-L3 OR VISTA interaction with its natural bindingpartner(s). Immune cells can be suboptimally stimulated in vitro with aprimary activation signal, for example, T cells can be stimulated withphorbol ester, anti-CD3 antibody or preferably, antigen, in associationwith an MHC class II molecule, and given a costimulatory signal, e.g.,by a stimulatory form of B7 family antigen, for instance by a celltransfected with nucleic acid encoding a B7 polypeptide and expressingthe peptide on its surface, or by a soluble, stimulatory form of thepeptide. The cells can then be contacted with cells expressing PD-L3 ORVISTA (e.g., antibodies against PD-L3 OR VISTA Known cytokines releasedinto the media can be identified by ELISA or by the ability of anantibody which blocks the cytokine to inhibit immune cell proliferationor proliferation of other cell types that are induced by the cytokine.For example, an IL-4 ELISA kit is available from Genzyme (Cambridge,Mass.), as is an IL-7 blocking antibody. Blocking antibodies againstIL-9 and IL-12 are available from Genetics Institute (Cambridge, Mass.).The effect of stimulating or blocking PD-L3 OR VISTA activity or theinteraction of PD-L3 OR VISTA and its binding partner(s) on the cytokineprofile can then be determined. As noted supra and shown in the examplesPD-L3 OR VISTA apparently suppresses the expression of IL-2 and gammainterferon by immune cells.

An in vitro immune cell costimulation assay as described above can alsobe used in a method for identifying novel cytokines which can bemodulated by modulation of PD-L3 OR VISTA activity. For example, wherestimulation of the CD28/CTLA4 pathway seems to enhance IL-2 secretion,stimulation of the ICOS pathway seems to enhance IL-10 secretion(Hutloff et al. (1999) Nature 397:263). If a particular activity inducedupon costimulation, e.g., immune cell proliferation, cannot be inhibitedby addition of blocking antibodies to known cytokines, the activity mayresult from the action of an unknown cytokine. Following costimulation,this cytokine can be purified from the media by conventional methods andits activity measured by its ability to induce immune cellproliferation.

To identify cytokines which may play a role the induction of tolerance,an in vitro T cell costimulation assay as described above can be used.In this case, T cells would be given the primary activation signal andcontacted with a selected cytokine, but would not be given thecostimulatory signal. After washing and resting the immune cells, thecells would be rechallenged with both a primary activation signal and acostimulatory signal. If the immune cells do not respond (e.g.,proliferate or produce cytokines) they have become tolerized and thecytokine has not prevented the induction of tolerance. However, if theimmune cells respond, induction of tolerance has been prevented by thecytokine. Those cytokines which are capable of preventing the inductionof tolerance can be targeted for blockage in vivo in conjunction withreagents which block B lymphocyte antigens as a more efficient means toinduce tolerance in transplant recipients or subjects with autoimmunediseases. For example, one could administer a cytokine blocking antibodyto a subject along with an agent that promotes PD-L3 OR VISTA activityor PD-L3 OR VISTA interaction with a binding partner.

Thus, to summarize a novel member of the Programmed Death Ligand (PDL)family has now been identified which is expressed by Treg cells. Thisnovel protein has been designated PD-L3 OR VISTA. The receptors of thisPD-L family are type I transmembrane proteins containing a single IgVdomain, while the ligands are type I transmembrane proteins expressingboth an IgV and an IgC extracellular domains. Like other members of thePDL family, PD-L3 OR VISTA co-stimulates αCD3 proliferation of T cellsin vitro. In addition, the expression of PD-L3 OR VISTA is increased inαCD3 activated Treg and reduced in the presence of αGITR.

A second, TNF-like, protein has also been identified as beingupregulated upon αCD3/αGITR stimulation. This protein has beendesignated Treg-sTNF. These proteins may be involved incontact-dependent and paracrine suppression of immunity and thereforeare useful for modulating (e.g., inhibiting or stimulating) an immuneresponse and in the treatment of diseases and conditions involving Tregsignaling. For example, the PD-L3 OR VISTA protein can be used as aco-stimulatory signal for stimulating or enhancing immune cellactivation. PD-L3 OR VISTA proteins and PD-L3 OR VISTA binding agentsand PD-L3 OR VISTA agonists and antagonists are especially useful intreating immune conditions wherein regulation of T cell immunity isdesired, e.g., modulation of T cell activation, differentiation andproliferation, and in particular modulation of CD4+ and CD8+ T cellproliferation, cytokine production, and T cell responses during cognateinteractions between T cells and myeloid derived APCs.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the Figures and Sequence Listing, areincorporated herein by reference.

EXAMPLES

The following Materials and Methods were used in the examples whichfollow: Materials and Methods

Expression Profiling

To facilitate comparisons with established expression profiles of Tregcells, standard growth and activation conditions were employed (McHugh,et al. (2002) supra). Briefly, fresh isolated Treg cells (˜96% positive)were inoculated at 106/mL into complete RPMI medium supplemented with10% fetal bovine serum and 100 units IL-2 in a 24-well plate precoatedwith anti-CD3 with or without anti-GITR (DTA-1)(Shimizu, et al. (2002)supra). The cells were cultured at 37° C. for 0 and 12 hours, RNA waspurified and subsequently analyzed using an Affymetrix® mouse genomeA430 oligonucleotide array.

By comparing the data from resting or activated CD4+CD25+ T cell groups,gene expression patterns were found to be similar to those establishedin the art (Gavin, et al. (2002) supra; McHugh, et al. (2002) supra). Toidentify genes regulated by GIRT signaling, gene expression profileswere compared between the different cell populations with or withoutanti-GITR treatment. A list of known as well as unknown genes werecompiled including the previously uncharacterized PD-L3 OR VISTA andTreg-sTNF.

Mice

C57BL/6 mice, and OTII CD4 transgenic mice were purchased from theJackson Laboratory. FoxP3-GFP reporter mice were as previously describedFontenot, J. D., Rasmussen, J. P., Williams, L. M., Dooley, J. L., Farr,A. G., and Rudensky, A. Y. (2005). Regulatory T cell lineagespecification by the forkhead transcription factor foxp3. Immunity 22,329-341 and were generously provided by Alexander Rudensky, Universityof Washington School of Medicine, Seattle, Wash. PD-1 KO mice weregenerously provided by Dr. Tasuku Honjo (Kyoto University, Japan)Nishimura, H., Nose, M., Hiai, H., Minato, N., and Honjo, T. (1999).Development of lupus-like autoimmune diseases by disruption of the PD-1gene encoding an ITIM motif-carrying immunoreceptor. Immunity 11,141-151; Nishimura, H., Okazaki, T., Tanaka, Y., Nakatani, K., Hara, M.,Matsumori, A., Sasayama, S., Mizoguchi, A., Hiai, H., Minato, N., andHonjo, T. (2001). Autoimmune dilated cardiomyopathy in PD-1receptor-deficient mice. Science 291, 319-322. All animals weremaintained in a pathogen-free facility at Dartmouth Medical School.

Abs, Cell Lines, and Reagent:

Antibodies αCD3 (2C11), αCD28 (PV-1), αCD4 (GK1.5), αCD8 (53-6.7),αCD11b (M1/70), αF4/80 (BM8), αCD11c (N418), αNK1.1 (PK136), αGr1(RB6-8C5), αPD-L1 (MINS), αPD-L2 (TY25), αB7-H3 (M3.2D7), αB7-H4 (188)were purchased from Ebioscience. LPS (Sigma), recombinant murine IFNPeprotech), human IL-2 (Peprotech), soluble PD-L1-Ig fusion protein (R&Dsystems) were used at indicated concentrations. Complete Freund'sadjuvant (CFA) and chicken ovalbumin (OVA) were purchased from Sigma.The CHO cell line expressing MHCII molecule I-Ad and costimulatorymolecule B7-2 was kindly provided by Dr. Arlene Sharpe (Harvard MedicalSchool).

Molecular Cloning of PD-L3 OR VISTA, Retrovirus Production andRetroviral Transduction of Cells

Full length PD-L3 OR VISTA was cloned from purified murine CD4+ T cells.Total RNA was isolated from CD4+ T cells using Qiagen RNAmini kit. cDNAwas generated using Bio-Rad iScript™ cDNA synthesis kit. Full-lengthPD-L3 OR VISTA was amplified and cloned into the ECorI-XhoI site of aretroviral vector pMSCV-IRES-GFP Zhang. X., and Ren, R. (1998). Bcr-Ablefficiently induces a myeloproliferative disease and production ofexcess interleukin-3 and granulocyte-macrophage colony-stimulatingfactor in mice: a novel model for chronic myelogenous leukemia. Blood92, 3829-3840, in which the IRES-GFP fragment was replaced by RFP, thusresulting in a fusion protein of PD-L3 OR VISTA fused to the N-terminusof RFP. Helper free retroviruses were generated in HEK293T cells bytransient transfection of the PD-L3 OR VISTA-RFP retroviral vectortogether with an ecotrophic packaging vector pCL-Eco (IMGENEX corp.).Retroviral transduction of murine T cell line EL4 cells, or bone marrowderived DCs were carried out by spin infection at 2000 rpm at RT for 45min in the presence of 8 μg/ml polybrene (Sigma).

Production of PD-L3 OR VISTA-Ig Fusion Protein

The extracellular domain of PD-L3 OR VISTA (amino acid 32-190) wasamplified and cloned into the SpeI-BamHI sites of the parental vectorCDM7B Hollenbaugh, D., Douthwright, J., McDonald, V., and Aruffo, A.(1995). J Immunol Methods 188, 1-7 . . . . This vector contains themutant form of constant and hinge regions of human IgG1, which has muchreduced binding to Fc receptors. The resulting vector CDM7B-PD-L3 ORVISTA was co-transfected with a DHFR expression vector pSV-dhfr (McIvor,R. S., and Simonsen, C. C. (1990)). Nucleic Acids Res 18, 7025-7032 intothe CHO (dhfr-) cell line (ATCC #CRL-9096). Stable CHO cell clones thatexpress PD-L3 OR VISTA-Ig were selected in medium MEM-alpha w/onucleotides (Invitrogen). Further amplification with 0.5-1 μMmethotrexate (Sigma M9929) yielded clones expressing high levels ofsoluble PD-L3 OR VISTA-Ig fusion protein. The fusion protein was furtherpurified from culture supernatant using standard protein-G columnaffinity chromatography.

Generation of PD-L3 OR VISTA Monoclonal Antibodies

Armenian hamsters were immunized 4× times with EL4 cells over-expressingPD-L3 OR VISTA-RFP weekly, then boosted with PD-L3 OR VISTA-Ig fusionprotein emulsified in CFA. Four weeks after the boost, hamsters wereboosted again with soluble PD-L3 OR VISTA-Ig fusion protein. Four daysafter the last boost, hamster spleen cells were harvested and fused tothe myeloma cell line SP2/0-Ag14 (ATCC #CRL-1581) using standardhybridoma fusion techniques Shulman, M., Wilde, C. D., and Kohler, G.(1978). A better cell line for making hybridomas secreting specificantibodies. Nature 276, 269-270. Hybridoma clones that secret PD-L3 ORVISTA specific antibodies were selected after limiting dilution andscreened by both ELISA and flow cytometric methods.

RNA and RT-PCR

Total RNA from various mouse tissue samples or purified hematopoieticcell types were collected by using Trizol™ (Invitrogen) method followingcompany's instructions. cDNAs were prepared by using the iScript™ cDNAsynthesis kit (Bio-Rad). Equal amount of tissue cDNAs (10 ng) were usedfor RT-PCR reactions to amplify full-length PD-L3 OR VISTA. PCR productswere viewed after running through a 1% agarose gel.

Flow Cytometry

Flow cytometry analysis was performed on FACSCAN using CellQuestsoftware (BD Bioscience). Data analysis was performed using FlowJosoftware (Treestar).

Cell Preparation

Total CD4+ T cells were isolated from naive mice using total CD4+ T cellisolation kit (Miltenyi). When indicated, enriched CD4+ T cells wereflow sorted into naïve (CD44low CD25-CD62Lhi) and memory (CD44hiCD25-CD62Llow) populations. For in vitro proliferation assays, CD4+ Tcells were labeled with 5 uM CFSE (Molecular Probes) for 10 min at 37 C,and washed twice before being stimulated.

In Vitro Plate-Bound T Cell Activation Assay

Purified CD4+ T cells (100,000 cells per well) were cultured in 96×flat-bottom well plates, in the presence of anti-CD3 (clone 2C11) andeither PD-L3 OR VISTA-Ig or control-Ig at indicated concentrationratios. For example, for a full-range titration, the 96-well plates werecoated with 2.5 μg/ml of αCD3 mixed together with 1.25 μg/ml (ratio2:1), 2.5 μg/ml (ratio 1:1), 5 μg/ml (ratio 1:2), or 10 μg/ml (ratio1:4) PD-L3 OR VISTA-Ig or control-Ig protein in PBS at 4° C. overnight.Wells were washed 3 times with PBS before adding CD4+ T cells. Replicatecultures were in complete RPMI 1640 medium supplemented with 10% FBS, 10mM HEPES, 50 μM β-ME, Penicillin/Streptomycin/L-Glutamine. Whenindicated, either 100 U/ml human IL-2 (PeproTech) or titrated amount of□CD28 (clone PV-1, Bio X cell) were coated together with □CD3 to rescuethe inhibitory effects of PD-L3 OR VISTA-Ig. Cultures were analyzed onday 3 for CFSE profiles, or according to a time course as indicated.

Culture of Bone Marrow Derived DCs, Retroviral Transduction, andStimulation of Transgenic CD4+ T Cells

Bone marrow derived DCs were generated as described Lutz, M. B.,Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., andSchuler, G. (1999). An advanced culture method for generating largequantities of highly pure dendritic cells from mouse bone marrow. JImmunol Methods 223, 77-92; Son, Y. I., Egawa, S., Tatsumi, T.,Redlinger, R. E., Jr., Kalinski, P., and Kanto, T. (2002). A novelbulk-culture method for generating mature dendritic cells from mousebone marrow cells. J Immunol Methods 262, 145-157 with somemodifications. Briefly, on day 0, bone marrow cells were isolated fromtibia and femur by flushing with 27 G needle. After red blood celllysis, 1-2×106 bone-marrow cells were resuspended in 1 ml complete RPMI1640 medium containing 20 ng/ml GM-CSF (Peprotech Inc), in 6× well cellculture plates (Nunc, Inc.). 2 ml supernatant containing either RFP orPD-L3 OR VISTA-RFP retrovirus was added to the bone marrow cells.Polybrene (Sigma) was also added at a final concentration 8 μg/ml.Infection was carried out by spinning the plate at 2000 rpm for 45 minat RT. Cells were then cultured for another 2 hours before fresh mediumwere added. Similar infection procedure was repeated on day+1, day+3,day+5, and dya+7. Loosely adherent cells (90% are CD11c+) were collectedon day+10 and CD11c+ RFP+ double positive cells were sorted and used tostimulate transgenic OT-II CD4+ T cells. For OT-II T cell proliferationassays, 100,000 CFSE-labeled OT-II CD4+ T cells were cultured in 96 wellround-bottom plates with 30,000 sorted RFP+ or PD-L3 OR VISTA-RFP+BMDCs, in the presence of titrated amount of synthetic OVA323-339peptide (Anaspec). Proliferation of OT-II T cells were analyzed at 72hrs by examining CFSE profiles.

Expression Studies of PD-L3 OR VISTA in Response to Immunization

To immunize transgenic mice D011.10, 300 μg OVA (Sigma) were emulsifiedin CFA (200 μl), and injected subcutaneously into the flanks of mice.The draining and non-draining inguinal lymph nodes were harvested atindicated time points. Single cell suspensions were prepared andanalyzed for the expression of PD-L3 OR VISTA and other surface markersby flow cytometry.

Inhibitory Activity of PD-L3 OR VISTA.

The inhibitory activity of PD-L1 was revealed by using antigenpresenting cells over-expressing PD-L1 in vitro with CD4+ and CD8+ Tcell antigen receptor transgenic T cells and antigen stimulation(Carter, et al. (2002) Eur. J. Immunol. 32:634-43). Similarly, thetentivector disclosed herein, which expresses the full-length PD-L3 ORVISTA, is transduced into cell lines expressing class II majorhistocompatibility complex (MHC) and class I MHC. The response of TEa Tgor the 2C transgenic T cells to antigen presented by emptyvector-transduced or PD-L3 OR VISTA-transduced antigen presenting cellsis determined according to established methods.

Protein Expression. Expression patterns in lymphoid, monocyte anddendritic cell subsets, as well as non-hemoatopoietic tissues, isdetermined by RT-PCR and western blot analysis using standard protocolsin combination with the rabbit αPD-L3 OR VISTA antibody disclosedherein.

Monoclonal Antibody Production. PD-L3 OR VISTA was overexpressed in themurine B cell line A20, and the recombinant cell line was used toimmunize Armenian hamsters. After 5× cell immunization, hamsters wereboosted with purified PD-L3 OR VISTA-Ig fusion protein emulsified inCFA. Four weeks later, a final boost was provided with soluble PD-L3 ORVISTA-Ig. Subsequently, fusions of hamster splenocytes with SP2/0 cellswere performed on day 4. Sixteen different clones were identified thatrecognized PD-L3 OR VISTA-Ig fusion protein by ELISA, as well as stainedPD-L3 OR VISTA but not PD-L1 overexpressed on the murine T cell lineEL4. Eleven of the clones were successfully subcloned and prepared forevaluation of their ability to stain endogenous PD-L3 OR VISTA on cellsand tissues, and to block PD-L3 OR VISTA functions.

Proliferation Assays:

In vitro CD4 T cell proliferation assays was designed to screen PD-L3 ORVISTA mAb activity. In this assay, T cells were stimulated byimmobilized anti-CD3 in microplate wells, which crosslinks T cellreceptors. Using a PD-L3 OR VISTA-1g fusion protein, which is composedof the extracellullar domain of PD-L3 OR VISTA fused to the Fc portionof human IgG, the activity of PD-L3 OR VISTA mAb was detected in twodifferent configurations. First, when mAb was co-immobilized with αCD3,it potently inhibited T cell proliferation, only in the presence ofadded soluble PD-L3 OR VISTA-Ig fusion protein. This activity wasdependent upon the ability of PD-L3 OR VISTA-Ig to bind to theimmobilized mAb in the well. Using this form of the assay, clones wereidentified that were of high, intermediate or low suppressive activity.Second, when mAb was added as a soluble reagent to the assay, it exertedpotent suppressive activity on T cell proliferation, by synergizing withthe immobilized PD-L3 OR VISTA-Ig fusion protein. In this form of assay,clones were identified that were of various suppressive activities.

Example 1: Cloning and Sequence Analysis of PD-L3 OR VISTA

PD-L3 OR VISTA and Treg-sTNF were identified by global transcriptionalprofiling of resting Treg, Treg activated with αCD3, and Treg activatedwith αCD3/αGITR. αGITR was selected for this analysis as triggering ofGITR on Treg has been shown to extinguish their contact-dependentsuppressive activity (Shimizu, et al. (2002) supra). PD-L3 OR VISTA andTreg-sTNF were identified on AFFIMETRIX® DNA arrays based on theirunique expression patterns (Table 1). PD-L3 OR VISTA exhibited anincrease in expression in αCD3 activated Treg and reduced expression inthe presence of αGITR; and Treg-sTNF exhibited a αCD3/αGITR-dependentincrease in expression.

Purified CD4+CD25+ T cells were stimulated in culture overnight withnone, αCD3, or αCD3/αGITR, and RNA isolated for real-time PCR analysis.Expression listed is relative to actin.

TABLE 1 Relative Expression mRNA None αCD3 αCD3/αGITR PD-L3 OR VISTA 610 7 T^(reg)-sTNF 0.2 0.3 1.5

Affymetrix analysis of activated vs. resting CD25+ CD4+ nTregs revealedthe expression of a gene product (RIKEN cDNA 4632428N05, or4632428N05Rik) with unknown function but with sequence homology to theIg superfamily.

More specifically, a 930 bp gene product was cloned from the CD4+ T cellcDNA library, which matched the predicted size and sequence.Silico-sequence and structural analysis predicts a transmembrane proteinof 309 amino acids upon maturation, with an extracellular domain of 159amino acids, a transmembrane domain of 22 amino acids and a cytoplasmictail of 95 amino acids (FIG. 1A). Amino acid sequence alignment revealsan extracellular Immunoglobulin (Ig)-V like domain homologous to B7family ligands such as PD-L1, PD-L2, B7-H3 and B7-H4, as well as to theB7 family receptors (i.e. PD-1, CTLA-4, CD28, BTLA, ICOS) (FIG. 1B-C).Although the sequence identity of the Ig-V domains between B7 familyligands and receptors in general is not very high (<40%), the Ig-Vdomain of 4632428N05Rik bears the highest homology with B7 familyligands PD-L1 and PD-L2. Sequence alignment also reveals several highlyconserved cysteines (FIG. 1B) that are important for intra-chaindisulfide bond formation, which is characteristic of the B7 familyligands Sica et al., (2003). Immunity 18, 849-861.

The extracellular domain of 4632428N05Rik contains only the Ig-V domainbut lacks the 1g-C domain (FIG. 1B-C). This unique feature ischaracteristic of the B7 family receptors, and distinguishes4632428N05Rik from all other B7 family ligands, which contain both Ig-Vand Ig-C domains Freeman, G. J. (2008). Proc Natl Acad Sci USA 105,10275-10276; Lazar-Molnar et al., (2008). Proc Natl Acad Sci USA 105,10483-10488; Lin et al., (2008), Proc Natl Acad Sci USA 105, 3011-3016;Schwartz et al., (2001), Nature 410, 604-608; Stamper et al., (2001),Nature 410, 608-61. Consistently, the phylogenic analysis using PhyMLalgorithm (Phylogenetic Maximum Likelihood) placed 4632428N05Rik in acloser evolutionary distance with B7 family receptors, in particularwith PD-1, than the B7 family ligands (FIG. 2) Guindon, S., and Gascuel,0. (2003). A simple, fast, and accurate algorithm to estimate largephylogenies by maximum likelihood. Syst Biol 52, 696-704. However, thecytoplasmic tail of PD-L3 OR VISTA does not contain any signalingdomains (e.g. ITIM, ITAM or ITSM), which are the signature domains of B7family receptors Sharpe, A. H., and Freeman, G. J. (2002). The B7-CD28superfamily. Nat Rev Immunol 2, 116-126. It is therefore hypothesizedthat despite its close evolutionary relationship with the inhibitoryreceptor PD-1, 4632428N05Rik represents a novel member of the B7 ligandfamily. Based on these structural and phylogenic characteristics, thismolecule was named PD-1-eXpressed as Ligand (PD-L3 OR VISTA). PD-L3 ORVISTA is also highly conserved between the mouse and human orthologs,sharing 77% sequence identity (FIG. 1D).

The nucleic acid sequence encoding mouse PD-L3 OR VISTA is set forthherein as SEQ ID NO:1 and the mouse PD-L3 OR VISTA protein sequence isset forth as SEQ ID NO:2.

The human homolog of PD-L3 OR VISTA is located on chromosome 10 (72.9Mb) and composed of 6 exons thereby generating a transcript of 4689bases in length coding for a 311 residue protein. The human homolog mRNAcoding sequence is provided in GENBANK accession number NM_022153 andprotein sequence give as NP_071436. The nucleic acid sequence encodinghuman PD-L3 OR VISTA is set forth herein as SEQ ID NO:3 and the humanPD-L3 OR VISTA protein sequence is set forth as SEQ ID NO:4. Mouse andhuman genes share 74% homology and are 68% identical at the proteinlevel. Homologs were also identified in Rattus norvegicus on chromosome20 (27.7 Mb; GENBANK accession number BC098723), as well as Fugurubripes and Danio rerio. In particular embodiments, PD-L3 OR VISTAproteins of the present share the common amino acid sequence set forthin SEQ ID NO:5.

Example 2: Expression Studies of PD-L3 OR VISTA by RT-PCR Analysis andFlow Cytometry

As shown in the experiments in FIG. 3, RT-PCR analysis was used todetermine the mRNA expression pattern of PD-L3 OR VISTA in mouse tissues(FIG. 3A). PD-L3 OR VISTA is mostly expressed on hematopoietic tissues(spleen, thymus, bone marrow), or tissues with ample infiltration ofleukocytes (i.e. lung). Weak expression was also detected innon-hematopoietic tissues (i.e. heart, kidney, brain, and ovary).Analysis of several hematopoietic cell types reveals expression of PD-L3OR VISTA on peritoneal macrophages, splenic CD11b+ monocytes, CD11c+DCs, CD4+ T cells and CD8+ T cells, but lower expression level on Bcells (FIG. 3B). This expression pattern is also largely consistent withthe GNF (Genomics Institute of Novartis Research Foundation) gene arraydatabase Su et al., (2002), Proc Natl Acad Sci USA 99, 4465-4470, aswell as NCBI GEO (gene expression omnibus) database (FIG. 4A-D).

In order to study the protein expression, PD-L3 OR VISTA specifichamster 8D8 and 6E7 monoclonal antibodies were produced. The specificityis demonstrated by positive staining on PD-L3 OR VISTA-overexpressingmurine EL4 T cells, but negative staining on PD-L1-overexpressing ELAcells (FIG. 5).

Both polyclonal and monoclonal antibodies were raised against PD-L3 ORVISTA. Using a rabbit anti-PD-L3 OR VISTA antibody. PD-L3 OR VISTAprotein was localized to lymphoid organs and prominently found in braintissue. Of the monoclonal antibodies identified, the specificity ofαPD-L3 OR VISTA clone 8D8 was further evaluated. In this analysis, clone8D8 was tested for binding against a panel of PD-L like-Ig fusionprotein molecules including CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2,PD-L3 OR VISTA and hlg. The results of this analysis indicated that 8D8αPDL-3 was highly specific for PD-L3 OR VISTA.

Specifically, using the anti-PD-L3 OR VISTA mAb clone 8D8, PD-L3 ORVISTA expression was analyzed on hematopoietic cells by flow cytometry.Foxp3GFP knock-in reporter mice were used to distinguish CD4+ nTregs(34). In peripheral lymphoid organs (spleen and lymph nodes),significant expression is seen on all CD4+ T cell subsets (see totalCD4+ T cells, or Foxp3-naïve T cells and Foxp3+ nTreg cells, and memoryCD4+ T cells), whereas CD8+ T cells express markedly lower amount ofsurface PD-L3 OR VISTA (FIG. 3C). In thymus, PD-L3 OR VISTA expressionis negative on CD4+CD8+ double positive thymocytes, low on CD4 singlepositive cells, and detectable on CD8 single positive cells. Next, astrong correlation of high PD-L3 OR VISTA expression with CD11b markercan be seen for both splenic and peritoneal cells, including both F4/80macrophages and myeloid CD11c+ DCs (FIG. 3D-E). On the other hand, Bcells and NK cells are mostly negative for PD-L3 OR VISTA expression. Asmall percentage of Gr-1+ granulocytes also express PD-L3 OR VISTA (FIG.3F).

A differential expression pattern is shown on the same lineage of cellsfrom different lymphoid organs (FIG. 3G). For CD4+ T cells and CD11bintermediate monocytes, the expression level follows the pattern ofmesenteric lymph node>peripheral LN and spleen>peritoneal cavity andblood. This pattern is less pronounced for CD11bhi cells. This datasuggests that PD-L3 OR VISTA expression on certain cell types might beregulated by cell maturity and/or tissue microenvironment.

In addition to freshly isolated cells, PD-L3 OR VISTA expression wasanalyzed on splenic CD4+ T cells, CD11bhi monocytes and CD11c+ DCs uponin vitro culture with and without activation (FIG. 6). Spleen cells wereeither cultured with medium, or with anti-CD3 (for activating T cells),or with IFN□ and LPS (for activating monocytes and DCs) for 24 hrsbefore being analyzed for the expression of PD-L3 OR VISTA and other B7family ligands (e.g. PD-L1, PD-L2, B7-H3 and B7-H4). This comparisonrevealed distinctive expression patterns between these molecules. PD-L3OR VISTA expression is quickly lost on all cell types upon in vitroculture, regardless of the activation status. In contrast, PD-L1expression is upregulated on CD4+ T cells upon stimulation, or onCD11bhi monocytes and CD11c+ DCs upon culture in medium alone, andfurther enhanced in the face of stimulation. The expression of PD-L2,B7-H3 and B7-H4 are not prominent under the culture conditions used. Theloss of PD-L3 OR VISTA expression in vitro is unique when compared toother B7 family ligands, but might reflect non-optimal cultureconditions that fail to mimic the tissue microenvironment.

To address how PD-L3 OR VISTA expression might be regulated in vivo, CD4TCR transgenic mice D011.10 were immunized with the cognate antigenchicken ovalbumin (OVA) emulsified in complete Freund's adjuvant (CFA).At 24 hrs after immunization, cells from the draining lymph node wereanalyzed for PD-L3 OR VISTA expression (FIG. 7A). Immunization withantigen (CFA/OVA) but not the adjuvant alone drastically increased theCD11b+ PD-L3 OR VISTA+ myeloid cell population, which contained a mixedpopulation of F4/80+ macrophages and CD11c+ DCs. Further comparison withPD-L1 and PD-L2 reveals that even though PD-L1 has the highestconstitutive expression level, PD-L3 OR VISTA is the most highlyupregulated during such an inflammatory immune response (FIG. 7B).Collectively, these data strongly suggest that the expression of PD-L3OR VISTA on myeloid APCs is tightly regulated by the immune system,which might contribute to its role in controlling immune responses andregulating T cell immunity.

In contrast to its increased expression on APCs, PD-L3 OR VISTAexpression is diminished on activated DO11.10 CD4+ T cells at a latertime point upon immunization (i.e. at 48 hr but not at 24 hr) (FIG. 8).This result suggests that PD-L3 OR VISTA expression on CD4 T cells invivo may be regulated by its activation status and cytokinemicroenvironment during an active immune response.

Example 3: Functional Impact of PD-L3 OR VISTA Signaling on CD4+ andCD8+ T Cell Responses

A PD-L3 OR VISTA-Ig fusion proteins were was produced to examine theregulatory roles of PD-L3 OR VISTA on CD4+ T cell responses. The PD-L3OR VISTA-Ig fusion protein contains the extracellular domain of PD-L3 ORVISTA fused to the human IgG1 Fc region. When immobilized on themicroplate, PD-L3 OR VISTA-Ig but not control Ig suppressed theproliferation of bulk purified CD4+ and CD8+ T cells in response toplate-bound anti-CD3 stimulation, as determined by arrested celldivision (FIG. 9A-B). The PD-L3 OR VISTA Ig fusion protein did notaffect the absorption of anti-CD3 antibody to the plastic wells, asdetermined by ELISA (data not shown), thus excluding the possibility ofnon-specific inhibitory effects. PD-1 KO CD4+ T cells were alsosuppressed (FIG. 9C), indicating that PD-1 is not the receptor for PD-L3OR VISTA. The inhibitory effect of PD-L1-Ig and PD-L3 OR VISTA-Ig wasalso directly compared (FIG. 10). When titrated amounts of Ig fusionproteins were absorbed to the microplates together with □CD3 tostimulate CD4+ T cells, PD-L3 OR VISTA-Ig showed similar inhibitoryefficacy as PD-L1-Ig fusion protein.

Since bulk purified CD4+ T cells contain various subsets, the impact ofPD-L3 OR VISTA-Ig on sorted naïve (CD25-CD44lowCD62Lhi) and memory(CD25-CD44hiCD62Llow) CD4+ T cell subsets was evaluated (FIG. 11). It isshown that PD-L3 OR VISTA can suppress the proliferation of bothsubsets, albeit with much less efficacy on the memory cells.

To further understand the mechanism of PD-L3 OR VISTA-mediatedsuppression, the expression of early TCR activation markers andapoptosis were measured following T cell activation in the presence orabsence of PD-L3 OR VISTA-Ig. Consistent with the negative impact oncell proliferation, there is a global suppression on the expression ofthe early activation markers CD69, CD44, and CD62L (supplemental FIG.12A). On the other hand, the PD-L3 OR VISTA-Ig fusion protein did notinduce apoptosis. On the contrary, less apoptosis (as determined by thepercentage of annexin V+ 7AAD− cells) was seen in the presence of PD-L3OR VISTA or VISTA-Ig than the control-Ig, at both early (24 hr) andlater stage (48 hr) of TCR activation (FIG. 12B). For example, at 24 hrtime point, on total “ungated” population, ˜27% cells were apoptotic inthe presence of PD-L3 OR VISTA or VISTA-Ig, but ˜39% control cells wereapoptotiC When examining the cells within the live cell R1 gate, it isapparent that PD-L3 OR VISTA or VISTA-Ig strongly inhibitedactivation-induced-cell-death (ACID), because about 72.6% control cellsbecame apoptotic whereas only 43.5% cells were apoptotic when treatedwith PD-L3 OR VISTA or VISTA-Ig. Similar results were seen for the 48 hrtime point. Therefore, it appears that PD-L3 OR VISTA or VISTAnegatively regulates CD4+ T cell responses by suppressing early TCRactivation and arresting cell division, but with minimum direct impacton apoptosis. This mechanism of suppression is similar to that of B7-H4Sica, G. L., Choi, I. H., Diu, G., Tamada, K., Wang, S. D., Tamura, H.,Chapoval, A. I., Flies, D. B., Bajorath, J., and Chen, L. (2003). B7-H4,a molecule of the B7 family, negatively regulates T cell immunity.Immunity 18, 849-861.

A 2-step assay was developed to determine whether PD-L3 OR VISTA orVISTA-Ig can suppress pre-activated CD4 T cells, and how persistent itssuppressive effect is. It is shown that the suppressive effect of PD-L3OR VISTA or VISTA-Ig fusion protein persists after its removal at 24 hrpost activation (FIG. 9D). In addition, both naïve and pre-activatedCD4+ T cells could be suppressed by PD-L3 OR VISTA or VISTA-Ig (FIG.9Di, 9Diii and 9Div).

Next, the impact of PD-L3 OR VISTA or VISTA-Ig on CD4+ T cell cytokineproduction was analyzed. PD-L3 OR VISTA or VISTA-Ig suppressed theproduction of Th1 cytokines IL-2 and IFNalpha from bulk purified CD4+ Tcell culture (FIG. 13A-B). The impact of PD-L3 OR VISTA or VISTA wasfurther tested on separate naïve (CD25-CD44lowCD62Lhi) and memory(CD25-CD44hiCD62Llow) CD4+ T cell populations. It is shown that memoryCD4+ T cells are the major source for cytokine production within theCD4+ T cell compartment, and PD-L3 OR VISTA or VISTA can suppress thisproduction (FIG. 13C-D). Similar inhibitory effect of PD-L3 OR VISTA orVISTA on IFNalpha production from CD8+ T cells was also shown (FIG.13E). This inhibitory effect of PD-L3 OR VISTA or VISTA on cytokineproduction by CD4+ and CD8+ T cells is consistent with the hypothesisthat PD-L3 OR VISTA or VISTA is an inhibitory ligand that down-regulatesimmune responses.

Next, studies were designed to determine the factors that are able toovercome the inhibitory effect of PD-L3 OR VISTA or VISTA. Given thatPD-L3 OR VISTA or VISTA suppressed IL-2 production, and IL-2 is criticalfor T cell survival and proliferation, we hypothesize that IL-2 mightcircumvent the inhibitory activity of PD-L3 OR VISTA or VISTA. As shownin FIG. 14A, exogenous IL-2, but not IL-15, IL-7, or IL-23, partiallyreversed the suppressive effect of PD-L3 OR VISTA or VISTA-Ig on cellproliferation. The incomplete rescue by high levels of IL-2 indicatesthat PD-L3 OR VISTA or VISTA signaling targets broader T cell activationpathways than simply IL-2 production. On the other hand, potentco-stimulation signal provided by anti-CD28 agonistic antibodycompletely reversed PD-L3 OR VISTA or VISTA-Ig mediated suppression(FIG. 14B), whereas intermediate levels of costimulation is stillsuppressed by PD-L3 OR VISTA or VISTA signaling (FIG. 14C). This resultsuggests that PD-L3 OR VISTA or VISTA-mediated immune suppression wouldbe more effective under less inflammatory conditions, but wilt beinevitably overwhelmed by strong positive costimulatory signals. In thisregard, PD-L3 OR VISTA or VISTA shares this feature with othersuppressive B7 family ligands such as PD-L1 and B7-H4 Sica et al.,(2003), Immunity 18, 849-861; Carter et al., (2002), Eur J Immunol 32,634-643.

In addition to PD-L3 OR VISTA or VISTA-1g fusion protein, it isnecessary to confirm that PD-L3 OR VISTA or VISTA expressed on APCs cansuppress antigen-specific T cell activation during cognate interactionsbetween APCs and T cells. For this purpose, PD-L3 OR VISTA or VISTA-RFPor RFP control protein was over-expressed via retroviral transduction inan artificial antigen presenting cell line (CHO-APC) that stablyexpresses MHCII and B7-2 molecules Latchman et al., (2001), Nat Immunol2, 261-268. One problem in expressing PD-L3 OR VISTA or VISTA in CHO isthat the majority of PD-L3 OR VISTA or VISTA failed to localize to thecell surface, perhaps due to the alien environment that lacks supportfor PD-L3 OR VISTA or VISTA surface localization (data not shown).Although there are no clear motifs present on the cytoplasmic tail ofPD-L3 OR VISTA or VISTA to suggest the mode of regulation, we speculatethat the tail might play a role for its intracellular localization.Consequently, a tail-less PD-L3 OR VISTA or VISTA mutant was designedand was found to successfully localize to CHO cell surface (data notshown).

To stimulate T cell response, CHO-PD-L3 OR VISTA or VISTA or CHO-RFPcells were incubated together with DO11.10 CD4+ T cells in the presenceof antigenic OVA peptide. As shown in FIG. 15 A-C, CHO-PD-L3 OR VISTA orVISTA induced less proliferation of DO11.10 cells than CHO-RFP cells.This suppressive effect is more pronounced at lower peptideconcentrations, consistent with the notion that a stronger stimulatorysignal would overcome the suppressive impact of PD-L3 OR VISTA or VISTA.

In addition, the inhibitory effect of full-length PD-L3 OR VISTA orVISTA on natural APCs was confirmed. In vitro cultured bone marrowderived dendritic cells (BMDC) do not express high level of PD-L3 ORVISTA or VISTA (FIG. 16). PD-L3 OR VISTA or VISTA-RFP or RFP wasexpressed in BMDCs by retroviral transduction during the 10 day cultureperiod. Transduced cells were sorted to homogeneity based on RFPexpression. The expression level of PD-L3 OR VISTA or VISTA ontransduced DCs was estimated by staining with anti-PD-L3 OR VISTA orVISTA mab, and found to be similar to the level on freshly isolatedperitoneal macrophages, thus within the physiological expression range(FIG. 16). Sorted BMDCs were then used to stimulate OVA-specifictransgenic CD4+ T cells (OTII) in the presence of OVA peptide (FIG.15D). It is shown therein that the expression of PD-L3 OR VISTA or VISTAon BMDCs suppressed the cognate CD4+ T cell proliferative responses.This result is consistent with previous data using PD-L3 OR VISTA orVISTA-Ig fusion protein and CHO-APC cells, suggesting that PD-L3 ORVISTA or VISTA can suppress T cell-mediated immune responses.

Example 4: Evaluation of Anti-PD-L3 OR VISTA or VISTA Antibodies inMultiple Sclerosis Animal Model (EAE)

Because the □PD-L3 OR VISTA or VISTA mAbs in vivo appeared to suppress Tcell responses, □PD-L3 OR VISTA or VISTA was tested to evaluate if itcan inhibit a T cell-mediated autoimmune disease. Using the ExperimentalAllergic Encephalomyelitis (EAE) model, the functional impact of □PDL-L3mAbs on inflammatory diseases was determined. EAE is a widely usedmurine model of the human autoimmune disease multiple sclerosis. EAE canbe induced by either immunization with myelin antigens in adjuvant or byadoptive transfer of myelin-specific T cells, which results ininflammatory infiltrates of various effector T cells and B cells, andmacrophages, and demyelination of central nervous systems.

αPDL-L3 mAb was tested in the passive EAE model to avoid induction ofanaphylaxis due to the injection of large amount of mAb as foreignantigen. In this adoptive transfer EAE model, donor SJL mice wereimmunized with CFA and PLP peptide. On day 10, total lymphocytes fromdraining LN were isolated, and cultured in vitro with PLP peptide, IL-23(20 ng/ml) and anti-IFNg (10 μg/ml) for 4 days. Expanded CD4 T cellswere then purified and adoptively transferred into naïve recipient mice.This analysis indicated that αPDL-L3 mAb delayed disease onset, as wellas reduced disease severity, thereby shifting the disease progressioncurve significantly (FIG. 17). In addition, it reduced severity in alarge percentage of the mice and greatly increased survival from around22% to over 75%. This demonstrated activity of αPDL-L3 mAb in EAE isconsistent with the in vitro data, and demonstrates the use of thisreagent as a novel immunoregulatory reagent in various inflammatorydiseases.

Example 5: Expression of Vista in the CNS

The expression of VISTA in the CNS was also effected. These assaysrevealed that in mice with disease, VISTA expression is markedly reduced(from 76%→33%) on the CD11b+ cells (FIG. 23), consistent with thehypothesis that the loss of VISTA may be permissive for enhancedinflammation. This is interesting, and likely functionally importantwhen we contrast inflammatory myeloid cells herein, with the MDSC intumors that express extremely high levels of VISTA. It has been reportedthat EAE mice have elevated numbers of myeloid derived suppressor cells(CD11b+Ly-6Chigh MDSC) in the spleen which are potently suppressive forT cell activation and may temper disease32. Our data strongly supportthe hypothesis that VISTA may play a role in myeloid-mediatedsuppression in EAR

Example 6: The Impact of VISTA on the Fate and Function of T Cells inEAE

We also conducted experiments assaying the effect of VISTA on the fateand function of T cells in EAE. We wanted to assess if VISTA alters thedevelopment of pathogenic, encephalitogenic T cells, clonal T cellexpansion, T cell polarity, longevity, and conversion of Teff→Treg. Westudied the impact of VISTA blockade on T cell fate in EAE. Consistentwith the higher disease score, analysis of CNS at the end of diseasecourse confirmed significantly more IL17A-producing CD4+ T cellinfiltration (from 0.66→11%) in 13F3 (□ VISTA) treated group (FIG. 24).

Example 7: PD-L3 OR VISTA or VISTA Transgenic and Knock-Out Mice

Using Lentiviral infection of embryos, four transgenic mice ubiquitouslyexpressing PD-L3 OR VISTA or VISTA have been produced. These miceexpress full-length PD-L3 OR VISTA or VISTA under the control of thehuman elongation factor 1 promoter. These mice were generated usinglentiviral vector pWPT. Similar to other PD-L1 family members (Appay, etal. (2002) J. Immunol. 168:5954-8), it is contemplated that PD-L3 ORVISTA or VISTA will function as a negative regulator in vivo whilefunctioning to co-stimulate αCD3 T cell proliferation in vitro. In thisrespect, these mice are expected to spontaneously develop autoimmunityand in vivo immune responses in the PD-L3 OR VISTA or VISTA transgenicmice (i.e., humoral immune responses, T cell priming, etc.) areevaluated to assess systemic autoimmune disease development.

For knock-out mice, PD-L3 OR VISTA or VISTA is inactivated by homologousrecombination. A BAC clone containing full-length PD-L3 OR VISTA orVISTA sequence was purchased from INVITROGEN™ (Carlsbad, Calif.). APD-L3 OR VISTA or VISTA targeting vector was generated by inserting a1.6 kb fragment located at the 5′ side of the second exon of PD-L3 ORVISTA or VISTA gene upstream the neomycin gene and the 5 kb fragmentlocated at the 3′ side of the third exon of PD-L3 OR VISTA or VISTA genedownstream the neomycin gene. B6-derived embryonic stem (ES) cells areelectroporated with PD-L3 OR VISTA or VISTA targeting vector andrecombined clones are selected. Selected clones are then injected intoC57BL/6 blastocysts and the resulting chimeric male offspring are matedto FLP-deleter mice to remove the neomycin cassette. Transmission of thetargeted allele in the offspring is determined by PCR from genomic DNA.The second and the third exon contain the PD-L3 OR VISTA or VISTAdomain, therefore, the resulting mice have only the inactivated form ofthe PD-L3 OR VISTA or VISTA molecule.

The overall immune capacity of PD-L3 OR VISTA or VISTA deficient mice isdetermined as with other PD-L−/− mice, including assessment of T cellresponses to antigen, humoral immune responses, overt autoimmunity(e.g., Systemic Lupus Erythematosus, inflammatory bowel disease), andincreased susceptibility to induced autoimmune disease (experimentalautoimmune encephalomyelitis) (Chen (2004) supra).

Example 8: PD-L3 OR VISTA or VISTA Specific Antibodies Tested inCollagen-Induced Arthritis Animal Model

As shown in the experiments in FIG. 18, male DBA/1J mice were immunizedat the base of their tail with 100 μl of emulsion containing 100 μgchick type-II collagen (C-II) in CFA (Mycobacterium tuberculosis 3.5mg/ml) and boosted IP with 100 μg aqueous C-II on day 21post-immunization. Mice of each treatment group (n=6) were eitheruntreated (NT-black circles), injected with 300 μg hamster IgG (HamIg-black squares) or injected with 300 μg of monoclonal-antibody “7c9”(red triangle) or “13F3” (green triangle), as indicated. Injections weregiven every 2 days. Arthritic swelling was scored on a scale of 0-4 foreach paw of each mouse on the days indicated. The arthritis score shownis the total score of all paws of mice in each treatment group dividedby the number of mice in the group.

Example 9: VISTA Blockade by a Specific VISTA Monoclonal AntibodyEnhances T Cell Responses In Vitro

A VISTA-specific mab (13F3) was identified which neutralizesVISTA-mediated suppression (FIG. 19). CD11b^(hi) myeloid APCs werepurified from naïve mice to stimulate OT-II transgenic CD4⁺ T cells inthe presence or absence of 13F3. Consistent with its neutralizingeffect, 13F3 enhanced T cell proliferation stimulated by CD11b^(hi)myeloid cells, which were shown to express high levels of VISTA.

Example 10: Anti-VISTA Enhances Anti-Tumor Immunity

Because of the capacity of anti-VISTA to enhance T cell activation, weassessed whether anti-VISTA would enhance the protective immune responseto an immunogenic tumor. A model in which we have a great deal ofexperience is the bladder carcinoma, MB49. MB49 expresses male antigen,and thus it is modestly immunogenic in female mice, although, it willgrow and kill female mice if there is no immune intervention. To testthe efficacy of □VISTA therapy, female mice were administered MB49 tumorcells subcutaneously (sq) and treated with □VISTA. Days thereafter, thesize of the tumor was measured until the mice had to be euthanized. Ascan be readily seen in FIG. 20 anti-VISTA therapy greatly impairs tumorgrowth. We believe that this is due to the ability of anti-VISTA tointensify cell-mediated immune (CMI) responses.

Example 11: Effect of 1: VISTA on Tumor Regression in 4 Murine TumorModels

Experiments in the immunogenic bladder carcinoma tumor MB49 have shownthat neutralization of VISTA using mab 13F3 and protects host from tumorgrowth. The data indicates that VISTA has a considerable negativeimmunoregulatory role in the microenvironment of a tumor because of itsextremely high expression of MDSCs. Studies examining the effect ofanti-mouse VISTA on the growth of immunogenic (MB49) and highlynon-immunogenic (B16) tumor models will further confirm the efficacy ofαVISTA therapy, shed light on the mechanism of action, and provide thebasis for selecting the optimal dose and timing. The rationale for eachtumor model is detailed below.

Tumor Name Tumor Type Host Groups ASSAYS MB49 Bladder B6 Female □VISTATumor growth Carcinoma Control Ig Survival MB49 Bladder B6 Male Immune/Carcinoma Autoimmmune B16.F10 Melanoma B6 Male or Assays female ID8Ovarian B6 Female Cancer

MB49 in female mice: We have already shown efficacy in this murinemodel. MDSCs in this model also express elevated levels of VISTA (notshown). In this model, due to the presence of H-Y antigen, the MB49tumor is modestly immunogenic. Since we know anti-VISTA therapy iseffective, we will use this model as a “positive” control to determinedosing (1-100 ug/mouse; and timing (day of tumor inoculation, or 4, 7,10 days after tumor; therapeutic intervention) of anti-VISTA therapy.

MB49 in male mice: Using doses and timing effective in female mice, theefficacy of anti-VISTA therapy in male mice (in which the tumor is lessimmunogenic) is determined.

B16 melanoma: Anti-CTLA-4 mab was shown highly effective in this model,and represents a non-immunogenic tumor where the mouse model has beenvaluable for predicting success in humans. Dosing regimes and timingwill be similar to those shown to be effective in the MB49 model.

ID8 Ovarian carcinoma: It is in this model, that VISTA expression hasbeen shown to be extremely high on MDSCs. Mice bearing ID8 tumor aretreated with αVISTA at the time of tumor inoculum or at day 5, 15, 25post inoculation.

Methods. B6 WT mice are used to determine the optimal dose and timing ofanti-VISTA treatment for the remission of all murine tumor models noted.The models to be used are listed in the above table.

The readout for this dose and timing assay are tumor growth kinetics.For MB49 and B16 studies, all tumor studies are done via intradermal(i.d.) inoculation and therefore tumor size can be readily measured.Tumor measurements is collected every 2-3 days using a caliper. In eachof these models, the impact of anti-VISTA or control antibody will betested for its ability to slow tumor growth or facilitate tumorregression. Growth of ID8 will be followed using a luciferase transducedID8 and whole body imaging using an IVIS Workstation. In addition, hostsurvival will also be determined.

Data on tumor growth is expressed as mean tumor volume ±SEM anddifferences between groups will be analyzed by two-tailed ANOVA.Probability (p) values less than 0.05 is considered statisticallysignificant. Survival data is analyzed using the Kaplan-Meier methodwith the Wilcoxon rank test and the log-rank test used to verify thesignificance of the difference in survival between groups. In the B16models, frequencies of mice that develop vitiligo is determined.

Using these methods slowed tumor growth and/or tumor regression in micetreated with anti-VISTA mAb is obtained as compared with mice treatedwith control ab in several of the non-immunogenic tumor models. It hasalready been shown that anti-VISTA treatment delays tumor growth in animmunogenic tumor model. As each of these tumor models have their ownspecific growth kinetics and, anticipated dependency on VISTA to confertumor growth and suppress immunity, mice will be administered mab eitherat the time of tumor inoculum or at times thereafter. Additionally, atleast 3 different concentrations of urn VISTA mab is tested to determinethe optimal dose for therapeutic benefit.

As shown in FIG. 21A-E, VISTA mab treatment reduced tumor growth in all4 of these tumor models wherein mice were inoculated either sq with A.MB49, B. MCA105, or C. EG7 tumor cells, or D. ip with ID8-luciferasetumor cells, and treated with VISTA mab 13F3 every other day (300 μg)beginning on day+1. Subcutaneous tumor growth was monitored. ForID8-luciferase tumor, mice were imaged on day 30 using Xenogen IVIS. E.VISTA expression on myeloid leukocytes in tumor-bearing mice was alsodetermined. Draining LN and tumor tissues (ascites) were analyzed forVISTA expression. These findings show that VISTA expressed on MDSC is amajor suppressive molecule that interferes with the development ofprotective anti-tumor immunity, and □VISTA relieves this suppressiveactivity allowing immune intervention and slowing growth of tumor. Thesefindings also support the conclusion that VISTA on myeloid cells inautoimmune disease plays a pivotal function in regulating the extent ofinflammation.

Example 12: Synthesis of Oligomeric VISTA and VISTA Fusion ProteinsUseful for the Treatment of Autoimmunity

Soluble VISTA-Ig in vitro is not suppressive nor can its binding tocells be readily detected. By contrast, this molecule bound to plasticis profoundly suppressive. In addition, studies using VISTA-Ig in vivodid not show overt activity (data not shown). With respect to thesestudies the VISTA-Ig that was created has mutations in the CH2-CH3domain precluding FcR binding, and therefore is not cytophilic in vivo.Recent studies have shown that tetrameric PD-L1 bound 100× higher (Kd6×10-8 M) than monomeric PD-L126 to PD-1, and that binding to cells wasreadily detectable. Tetrameric PD-L1 was not tested in vivo, but invitro it was shown to block the functional suppression by native PD-L1.Using similar methods oligomers are made that will target the VISTApathway and elicit potent immunosuppressive activity in vitro ad invivo.

Such oligomers are constructed using the monomeric extracellular domainof VISTA or a fragment thereof, e.g., at least 50, 75, 100, 125, 150,175 or 200 amino acids long which extracellular domain or a portionthereof is used as the building blocks for oligomer. In these methodsthe inventors take advantage of the well-established MHC tetramertechnologies. In these methods the VISTA ectodomain construct or afragment is linked to the N-terminus of a variety of oligomerizationdomains (identified supra) in order to generate a series of VISTAcomplexes with valencies that span from divalent to heptavalent.

Thereby, a series of non-covalent oligomers is created based on highaffinity coiled-coil domains that direct the stable formation ofdimeric, trimeric, tetrameric, pentameric and heptameric assemblie.These oligomeric constructs are expressed in a host cell, e.g., E. coli.When expression is effected in E. coli the expressed oligomers are thenrefolded and purified from inclusion bodies using standard laboratoryprotocols. This approach has routinely produced high quality materialfor biological and structural analysis, including MHC-peptide complexesand trimeric GITRL66. The isolated oligomeric proteins are then assessedby SDS-PAGE, analytical gel filtration, analytical ultracentrifugationand mass spectrometry. These quality control measures ensure theavailability of homogeneous, well-characterized materials for in vitroand in vivo studies. The parallel organization of these constructsresults in molecules in which the valency is equal to the oligomericstate since each individual VISTA complex is positioned to productivelyinteract with cell surface bound VISTA receptor. The above constructspossess extreme stability and homogeneitiy of oligomeric state.(Non-covalent coiled-coil oligomerization domains typically exhibitmelting temperatures that exceed 100° C., except for the heptamersequence which exhibits a melting temperature of 95° C.

In addition dimeric VISTA-Ig is tetramerized that is either cytophilicor not cytophilic. The Fc fusion constructs of VISTA in frame with theIgG1 Fc (both wild-type IgG1 and the existing non-FcR-binding IgG1) aremodified with an N-terminal BirA site for enzymatic biotinylation andcloned into the pIRES2-EGFP vector. Enzymatic biotinylation will allowspecific, single residue modification and orientation upon avidinmultimerization. This approach has been used for the generation ofnumerous Ig-fusion proteins, including B7-1, PD-L1, PD-L2 and TIM-3. Theexpressed proteins are then enzymatically biotinylated in vitro,purified by size exclusion HPLC, and tetramerized using PE-Avidin. Theresulting tetramers which are cytophilic or not, are assessed in vivo.

These engineered multimeric VISTA proteins are useful in treatingautoimmunity and other conditions wherein intervention in the VISTApathway and immunosuppression is therapeutically warranted.

Example 13: VISTA Adenoviral Vectors for Inducing Immune Suppression

Gene transfer using recombinant adeno-associated virus (AAV) has seengreat technological development in gene therapy Specifically,AAV-mediated gene delivery of PD-L1 gene, or CTLA4-Ig and CD40-Ig hasachieved therapeutic efficacy in autoimmune disease models of lupus andcardiac transplantation. These methods will be used to deliver eitherfull length VISTA, or oligomeric VISTA ectodomains, and theirtherapeutic effects are assessed in the EAE model. Recombinantadenovirus vector expressing either full-length murine VISTA, oroligomeric VISTA ectodomain, is created using the Adeno-XTM ExpressionSystem (Clontech) according to the manufacturer's instructions. Briefly,VISTA is cloned into an E1 and E3-deleted, pAdDEST-based expressionvector, under the control of the human cytomegalovirus (CMV) promoter.VISTA and control lacZ expressing adenovirus are then purified from celllysates. For systemic overexpression of VISTA, adenovirusis administeredto mice by intravenous tail vein injection (1×109 plaque-forming units[Pfu]) either prior to or shortly after disease induction viaimmunization, or after disease onset. The control mice will receive 100μl PBS. Disease development and alterations are monitored in both SJLmice and C57BL/6 mice, which exhibit different disease progressionpattern, and which represent two distinct forms of clinicalmanifestation of human MS patients.

Example 14: Functional Studies with Engineered Proteins and AdenoviralVectors

Mice are also administered (5-100 ug of protein/mouse×3 weekly) withengineered VISTA and/or adenoviral vectors. Following administration, Tcell expansion, differentiation, as well as EAE development isdetermined.

Example 15: Structural Studies on VISTA and Determining MolecularDeterminants of VISTA Function

Affinity, specificity, oligomeric state, and the formation andlocalization of organized signaling complexes are critical contributorsto immune function. All of these features impact signaling and immuneregulation, as the organization of the receptor-ligand ectodomainsdirectly controls the recruitment, organization and function ofnon-covalently associated cytoplasmic signaling and scaffoldingmolecules. The high resolution crystal structure of VISTA is determinedusing techniques including bacterial, insect and mammalian expressionsystems, as well as high-throughput crystallization and structuredetermination approaches. To validate the crystallographically-observeddisulfide bonding pattern, we will exploit high resolution massspectrometry using approaches that successfully supported our publishedstudies of TIM-3 and human DcR359. Based on these structural results, aseries of mutants with altered oligomeric properties is designed, aswell as mutants in the vicinity of any perturbed regions of the VISTAIgV domain. These mutant proteins will provide additional directmechanistic insight into VISTA function and should be useful intherapeutics wherein immunosuppression is desired such as theautoimmune, allergic and inflammatory diseases identified herein. Thesemutants, especially oligomers are tested in in vitro systems and areassessed in animal autoimmune and inflammatory disease models in orderto assess the immunosuppressive effect on disease progression, diseaseremission or in protecting the animal from developing the autoimmune orinflammatory condition.

These oligomeric VISTA proteins will activate the VISTA pathway andfunction as a target of immune intervention in autoimmunity. Thisintervention will suppress immunity and exert a therapeutic benefit onautoimmune disease and other conditions wherein autoimmune suppressionis desired. This is accomplished by administering the oligomerized VISTAproteins in different autoimmune and inflammatory models such as the EAEand collagen-induced arthritis animal models. In addition, as discussedabove, adenoviral vectors that over-express full-length VISTA or VISTAoligomers are constructed and tested in vivo. These studies will confirmthe immunosuppressive effects of VISTA oligomers.

Example 16: Experiments Using Conditional Over-Expressing VISTATransgenic Mouse Strain (VISTA Transgenic Mouse Strain: R26StopFLVISTA(VISTA)

A targeting construct containing the full-length cDNA of VISTA precededby a loxP-flanked STOP cassette, has been targeted into the ubiquitouslyexpressed ROSA26 locus. Multiple correctly targeted R26StopFL/− VISTApups were born, and bred onto the CMV-Cre deleter strain60. Preliminarydata in the VISTA×CMV-cre confirm GFP and heightened VISTA expression.Studies on the immune status of these mice (T cell responses to antigen,antibody titers, etc) will confirm a suppressed phenotype. The VISTAstrain will be interbred with CD4-cre, CD11c-cre, and Lys-Cre todetermine if the lineage location of VISTA expression influencessuppression. The phenotype and function of the T cells is alsodetermined and it is determined if over-expression of VISTA results inthe generation of aTreg. In these studies Tregs from OVA-immune cre xVISTA strain are adoptively transferred into WT hosts, to see if antigenimmunization in the presence of over-expressed VISTA inducesantigen-specific Tregs. This should verify that VISTA impacts Tregdifferentiation.

In addition, studies are effected in the EAE model whereby the impact ofVISTA proteins on different lineages (by interbreeding with CD4-,CD11c-, Lys-cre) with respect to disease development is assessed.Assuming that disease can be suppressed by lineage restrictedoverexpression of VISTA mutants or in the CMV×VISTA mutant the temporalcontrol of disease development is also using Cre-ERT2× VISTA□. Throughthe administration of tamoxifen we can induce overexpression of VISTAprior to, or at disease initiation or at peak disease to determine ifVISTA can impact on the induction and/or effector phases of immunity.Using BM chimeric mice, temporally-restricted overexpression of VISTAcan be restricted to the hematopoietic compartment. For an appreciationof controlling the window of time VISTA is overexpressed, VISTA isgenetically turned on, then serologically turned off with theadministration of anti-VISTA mab. These studies will determine where andwhen VISTA has to act to control the development and progression ofautoimmune disease.

Example 17: Effect of Anti-VISTA Antibodies CD40/TLR Agonist Vaccine

As shown in FIG. 22, experiments were conducted that assayed the effectof anti-VISTA antibodies on vaccine efficacy. These results show thatanti-VISTA enhances the therapeutic efficacy of a CD40/TLR vaccine.C57BL/6 mice were challenged with 1×105 metastatic B16.F10 melanomacells s.q. Four days later, mice were vaccinated with 100 μg of thetumor associated antigen ΔV, 100 μg αCD40 FGK45 (CD40 agonisticantibody) and 100 μg S-27609(TLR7 agonist) with or without anti-VISTA(200 ug×3/week). Growth of tumor was monitored by caliper measurements.

Having described the invention the following claims are provided. Theseclaims are intended to cover all generic and specific features describedherein, and all statements of the scope which, as a matter of language,might be said to fall there between.

1. An isolated, or recombinant immunosuppressive multimeric VISTAprotein or conjugate thereof that comprises at least of two copies of apolypeptide which is at least 90% identical to the extracellular domainof the human or murine VISTA polypeptide in SEQ ID NO:2 or 4 whichcontains at least two copies of a polypeptide which is at least 90%identical to a fragment of the extracellular domain of said VISTApolypeptide which is at least 50 amino acids along. 2-52. (canceled)